Reprogramming T cells and hematopoietic cells

ABSTRACT

Methods and compositions relating to the production of induced pluripotent stem cells (iPS cells) are disclosed. For example, induced pluripotent stem cells may be generated from CD34 +  hematopoietic cells, such as human CD34 +  blood progenitor cells, or T cells. Various iPS cell lines are also provided. In certain embodiments, the invention provides novel induced pluripotent stem cells with a genome comprising genetic rearrangement of T cell receptors.

This application is a continuation of U.S. application Ser. No.13/376,361, filed Feb. 16, 2012, now U.S. Pat. No. 8,741,648, which is anational phase application under 35 U.S.C. §371 of InternationalApplication No. PCT/US2010/037376, filed Jun. 4, 2010, which claimspriority to U.S. Application No. 61/184,546 filed on Jun. 5, 2009 andU.S. Application No. 61/240,116 filed on Sep. 4, 2009, the entiredisclosure of each of which are specifically incorporated herein byreference in their entirety without disclaimer.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of molecularbiology and stem cells. More particularly, it concerns reprogramming ofsomatic cells, especially T cells and hematopoietic cells.

2. Description of Related Art

In general, stem cells are undifferentiated cells which can give rise toa succession of mature functional cells. For example, a hematopoieticstem cell may give rise to any of the different types of terminallydifferentiated blood cells. Embryonic stem (ES) cells are derived fromthe embryo and are pluripotent, thus possessing the capability ofdeveloping into any organ or tissue type or, at least potentially, intoa complete embryo.

Induced pluripotent stem cells, commonly abbreviated as iPS cells oriPSCs, are a type of pluripotent stem cell artificially derived from anon-pluripotent cell, typically an adult somatic cell. Inducedpluripotent stem cells are believed to be identical to naturalpluripotent stem cells, such as embryonic stem cells in many respects,such as in terms of the expression of certain stem cell genes andproteins, chromatin methylation patterns, doubling time, embryoid bodyformation, teratoma formation, viable chimera formation, and potency anddifferentiability, but the full extent of their relation to naturalpluripotent stem cells is still being assessed.

IPS cells were first produced in 2006 (Takahashi et al., 2006) frommouse cells and in 2007 from human cells (Takahashi et al., 2007a; Yu etal, 2007). This has been cited as an important advancement in stem cellresearch, as it may allow researchers to obtain pluripotent stem cells,which are important in research and potentially have therapeutic uses,without the controversial use of embryos.

In humans, iPS cells are commonly generated from dermal fibroblasts.However, the requirement for skin biopsies and the need to expandfibroblast cells for several passages in vitro make it a cumbersomesource for generating patient-specific stem cells. Moreover, previousmethods for reprogramming of human somatic cells are inconvenientbecause they need to obtain somatic cells directly from a human subject,or maintain the cells in a labor-intensive cell culture system.Therefore, there is a need to develop methods to induce pluripotent stemcells from alternative sources which are simple, convenient, and easilyaccessible. In developing the present invention, the inventorsconsidered that blood samples may be such a source because blood may becollected from a patient or a healthy individual, stored or transferred,for example, from a central unit for distribution to one or more remoteplaces. However, there have been no reports in producing pluripotentstem cells from T cells from such a clinically accessible source untilthis application to the inventors' knowledge, demonstrating asubstantial need to develop such technologies.

SUMMARY OF THE INVENTION

The present invention overcomes a major deficiency in the art inproviding induced pluripotent stem cells derived from T cells and/orhematopoietic progenitor cells by reprogramming. The present methodscould produce iPS cells from a clinically accessible source of T cells,such as a 3 ml whole blood sample, circumventing the need ofmobilization of hematopoietic cells. In other embodiments, hematopoieticcells, such as human or mammalian CD34⁺CD45⁺CD43⁺ hematopoieticprecursor cells, may be obtained from a blood sample and converted toiPS cells. Hematopoietic precursor cells may be obtained from a bloodsample of peripheral blood, e.g., via enrichment of CD34⁺ cells ordepletion of non-CD34⁺ cell lineages. In certain embodiments, CD34⁺hematopoietic cells may be obtained from a blood sample, such as arefrigerated or cryopreserved blood sample, which was obtained withoutmobilizing CD34⁺ hematopoietic progenitor cells in the subject prior toobtaining the blood sample. In this way, iPS cells may be generated froma wide variety of blood samples, including peripheral blood samplesfound at blood banks.

Therefore, there are provided methods for producing induced pluripotentstem cells from T cells and/or hematopoietic progenitor cells comprisingthe steps of: (a) obtaining a cell population comprising T cells and/orhematopoietic progenitor cells; and (b) producing iPS cells from T cellsand/or hematopoietic progenitor cells of the cell population to providean iPS cell population. Exemplary sources of the cell population mayinclude, but are not limited to, blood samples, blood components, bonemarrow, lymph node, fetal liver, or umbilical cord. The source of thecell population may comprise a blood sample or cells derived from ablood sample, wherein the blood sample was obtained from a subjectwithout externally mobilizing hematopoietic progenitor cells in thesubject (e.g., via external administration of a hematopoietic growthfactor to the subject) prior to obtaining the blood sample.

The cell population may be obtained from a cryopreserved blood sample orthe source of cell population or the cell population may have beencryopreserved. It was demonstrated that a cryopreserved blood samplecould be used as a source of T cells for successful reprogramming intoiPS cells in the Examples.

A particular advantage of certain aspects of the present invention isthe ability to practice certain aspects of the present invention throughthe use of a small volume of blood samples. The suitable volume of ablood sample could be from about 1 to about 5 ml, about 1 to 10 ml,about 1 to 15 ml, or more specifically, about 3 ml.

Hematopoietic stem/progenitor cells, like CD34⁺ cells, may be inducedwith extrinsically applied G-CSF to mobilize into peripheral blood forenrichment in a peripheral blood source. It has been found in certainaspects of the present invention that peripheral blood cells fromnon-mobilized donors can achieve successful reprogramming, thereforemobilization of bone marrow cells by extrinsically applied growthfactors are not needed. Thus, in a particular aspect, the source of thecell population may be a subject whose cells have not been mobilizedwith one or more extrinsically applied hematopoietic growth factors,e.g., granulocyte colony-stimulating factor (G-CSF). The term“extrinsic” or “external,” as used interchangeably herein, refers toapplication of a mobilizing agent from outside the organism, in contrastto use of CD34⁺ cells that have been mobilized to some degree byintrinsic factors that originate from within the organism.

To provide a population of T cells suitable for reprogramming, the cellpopulation comprising T cells may be prepared under conditions that willactivate the T cells in vitro, such as in the presence of an anti-CD3antibody, or in vivo (and thus have a specific TCR for a particularantigen, e.g., a cancer antigen for melanoma such as GP-100). This mayalso include the use of tetramers, vaccines and/or in vitro peptidestimulations known in the art. The cell population may also be culturedin vitro with one or more cytokines (e.g., IL-2) to expand the T cellpopulation therein. The T cells may be human T cells. In a particularaspect, the T cells may be CD4⁺, CD8⁺ T cells, or a combination thereof.Non-limiting examples of T cells include T helper 1 (TH1) cells, Thelper 2 (TH2) cells, TH17 cells, cytotoxic T cells, regulatory T cells,natural killer T cells, naïve T cells, memory T cells, gamma delta Tcells and any T cells.

In certain aspects, the cell population comprises from about 80% toabout 99%, about 90% to about 99%, about 97% to about 99%, or anyintermediate range of T cells, which may correspond to from at least,about, or at most, 1×10³, 2×10³, 3×10³, 4×10³, 5×10³, 6×10³, 7×10³,8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴, 4×10³, 5×10⁴, 6×10⁴, 7×10⁴, 8×10⁴,9×10⁴, 1×10⁵, 2×10⁵, 3×10⁵, 4×10⁵, 5×10⁵, 6×10⁵, 7×10⁵, 8×10⁵, 9×10⁵,1×10⁶, 2×10⁶ T cells or any range derivable therein. For example, theinventors demonstrated reprogramming in 96 well plates with as little asapproximately 1-5×10³ T cells per well (FIGS. 6A-6B).

To provide a population of hematopoietic precursor cells, the cellpopulation comprising hematopoietic cells may be prepared underconditions that will result in an enrichment or expansion of CD34⁺cells. Specifically, the invention finds that mobilization of bonemarrow cells is not required for obtaining enough CD34⁺ cells forreprogramming. For example, magnetic activated cell sorting (MACS) orfluorescence activated cell sorting (FACS) may be used to enrich CD34⁺hematopoietic cells; in certain embodiments, an Indirect CD34 MicroBeadKit or a Direct CD34 MicroBead Kit (both available from Miltenyi Biotec,Bergisch Gladbach, Germany) may be used with MACS to enrich CD34⁺hematopoietic cells from a sample, such as a peripheral blood sample.Additional methods are also known in the art for obtaining mobilizedCD34⁺ hematopoietic progenitor cells from peripheral blood, includingthe methods described in Gratwohl et al. (2002). Nonetheless, in certainpreferred embodiments, the CD34⁺ hematopoietic precursor cells may beobtained from a subject which has not been exposed to one or morehematopoietic growth factors; thus, the CD34⁺ hematopoietic precursorcells may advantageously be obtained from a blood sample of a donor thathas not been mobilized by one or more extrinsically applied growthfactors, including blood samples typically found in blood banks. Inother embodiments, CD34⁺ cells may be enriched in a sample via depletionof mature-hematopoietic cells, such as T cells, B cells, NK cells,dendritic cells, monocytes, granulocytes, and/or erythroid cells. Forlineage depletion, the cell suspension may be incubated with a cocktailof antibodies (e.g., one or more of CD2, CD3, CD11b, CD14, CD15, CD16,CD19, CD56, CD123, CD235a) which may then be used to remove the abovementioned lineage positive cells (e.g., Karanu et al., 2003). TheLineage cell Depletion Kit (Miltenyi Biotec, Bergisch Gladbach, Germany)is also commercially available and may be used for this purpose. Incertain embodiments, a combination of SCF, Flt3L, and/or IL-3 cytokinesmay be used to expand and proliferate CD34⁺ cells prior to conversion toiPS cells, e.g., using the method described in Akkina et al. (1996) orStemPro™-34 media (available from Invitrogen, Carlsbad, Calif., USA).

It is anticipated by the inventors that virtually any hematopoieticprogenitor cell or CD34⁺ hematopoietic cell may be reprogrammed into aniPS cell via the methods described herein. In certain embodiments,hematopoietic precursor cells obtained or derived from a peripheralblood sample may be converted into iPS cells via the methods providedherein. The hematopoietic precursor cells may express both CD34 andCD45, or CD34, CD45, and CD43. In certain instances it may be desirableto generate hematopoietic precursors from stem cells such as humanembryonic stem cells (hESC); in these embodiments, CD34⁺CD43⁺CD45⁺hematopoietic cells highly enriched in myeloid progenitors may begenerated, e.g., by coculture of hESC with OP9 feeder cells as describedin Choi et al. (2009). In certain instances the hematopoietic precursorcells may be negative for CD34 (e.g., Guo et al., 2003); it isanticipated that these hematopoietic precursor cells may nonetheless bedifferentiated into iPS cells.

To produce iPS cells from T cells and/or hematopoietic progenitor cellsof the cell population, the methods may comprise introducing one or morereprogramming factors into the T cells and/or hematopoietic progenitorcells. In a certain aspect, the reprogramming factors may bereprogramming proteins comprising a Sox family protein and an Oct familyprotein, one or more or each of which may be operatively linked to aprotein transduction domain for cellular entry. In a further embodimentof the invention, the reprogramming factors may be encoded by one ormore expression cassettes, and may include, for example, a Sox familyprotein and an Oct family protein. Sox and Oct are thought to be centralto the transcriptional regulatory hierarchy that specifies ES cellidentity. For example, Sox may be Sox1, Sox2, Sox3, Sox15, or Sox18,particularly Sox2; Oct may be Oct4. Additional factors may increase thereprogramming efficiency, like Nanog, Lin28, Klf4, c-Myc, SV40 Large Tantigen, or Esrrb; specific sets of reprogramming factors may be a setcomprising Sox2, Oct4, Nanog and, optionally, Lin-28; or comprisingSox2, Oct4, Klf and, optionally, c-Myc.

In a particular embodiment, the one or more expression cassettes maycomprise one or more polycistronic transcription units. Thepolycistronic unit may comprise different combination of operably linkedcoding regions, for example, (i) at least two reprogramming genes, suchas Sox-Oct, c-Myc-Klf, or Nanog-Lin28; alternatively, (ii) areprogramming gene linked with a selectable or screenable marker. Theaspect (i) may be preferred because the inventors have found that byswitching to using bicistronic vectors that have two of thereprogramming factors per vector (Sox2 and Oct4, cMyc and Klf4, or Nanogand Lin28) without any fluorescent marker (vector maps are representedin FIGS. 11A-11C) instead of using four separate bicistronic vectorswith one reprogramming factor and a fluorescent marker (such a vectormap is represented in FIG. 10), the reprogramming efficiency of usingthese former vectors have improved dramatically and the iPS coloniescome up earlier (˜day 10-14 rather than day 20-24).

To co-express multiple gene in the same polycistronic transcriptionunit, the polycistronic transcription unit may comprise an internalribosome entry site (IRES) or a sequence coding for at least oneprotease cleavage site and/or self-cleaving peptide for polycistronictranscription. For example, the self-cleaving peptide is a viral 2Apeptide.

In a still further embodiment, the one or more expression cassettes arecomprised in a reprogramming vector selected from the group consistingof a viral vector, an episomal vector, or a transposon. Morespecifically, the vector may be a retroviral vector, such as murineleukemia virus (MLV), Moloney murine leukemia virus (MMLV), Akv-MLV,SL-3-3-MLV or another closely related virus. The viral vector could alsobe a lentiviral vector. In certain aspects, the transcriptionalregulatory element may comprise a long terminal repeat region (LTR) tomediate integration of viral genes.

In an alternative aspect, the vector may be an episomal vector, such asan EBV-based vector, or a transposon-based vector.

In a further embodiment, the reprogramming factors may be introduced byliposome transfection, nucleofection, electroporation, particlebombardment, calcium phosphate, polycation, or polyanion, or any methodssuitable for introducing exogenous elements into the cells.

In some further aspects, the iPS cells could be selected based on one ormore embryonic stem cell characteristics, such as an undifferentiatedmorphology, an embryonic stem cell-specific marker, an adherentproperty, pluripotency, multi-lineage differentiation potential or anycharacteristics known in the art. For example, it may be particularlyconvenient to select the progeny cells on the basis of theundifferentiated morphology. The embryonic stem cell-specific markercould be one or more specific markers selected from the group consistingof SSEA-3, SSEA-4, Tra-1-60 or Tra-1-81, Tra-2-49/6E, GDF3, REX1, FGF4,ESG1, DPPA2, DPPA4, and hTERT. This selection step may be employed atmore than one time points after reprogramming to ensure that cells arein a pluripotent state and does not return to a differentiated state.IPS cells are also different from the T cells and hematopoieticprogenitor cells in their adherent property to a surface, which couldalso be employed in a convenient separation method.

In a particular aspect, the iPS cells may be selected based onessentially no expression of introduced exogenous elements such asvector genetic elements or reporter genes comprised in the expressioncassettes, because a reprogrammed cell is able to silence exogenouslyintroduced material as a cell has become pluripotent. Therefore, anessential loss of integrating vector genetic elements, or reporterexpression, e.g., fluorescence, is an indication in addition tomorphological characteristics that cell has been reprogrammed. Forexample, the silence of reporter expression may be selected byfluorescence-activated cell sorting (FACS), CAT assay or luminescenceassay based on the reporter gene introduced. “Essential loss” or“essentially free” of exogenous elements means that less than 1%, 0.5%,0.1%, 0.05%, or any intermediate percentage of cells of an iPS cellpopulation comprises exogenous elements. The iPS cell population may beessentially free of integrated, exogenous viral elements.

For clinical application of the iPS cells, the methods may furthercomprise differentiating the iPS cells to a differentiated cell, forexample, a cardiomyocyte, a hematopoietic cell, a myocyte, a neuron, afibroblast, a pancreatic cell, a hepatocyte, or an epidermal cell. In afurther aspect, a differentiated cell, tissue or organ, which has beendifferentiated from the iPS cell population as described above may bedisclosed. The tissue may comprise nerve, bone, gut, epithelium, muscle,cartilage or cardiac tissue; the organ may comprise brain, spinal cord,heart, liver, kidney, stomach, intestine or pancreas. In certainaspects, the differentiated cell, tissue or organ may be used in tissuetransplantation, drug screen or developmental research to replaceembryonic stem cells.

In a still further aspect, an induced pluripotent stem cell producedaccording to the methods above may also be disclosed. There may also beprovided an induced pluripotent stem cell comprising a genome comprisingan incomplete set of V, D, and J segments of T cell receptor genescompared with an embryonic stem cell, which may be a human cell. In aparticular aspect, the induced pluripotent stem cell may be essentiallyfree of integrated, exogenous viral elements.

Embodiments discussed in the context of methods and/or compositions ofthe invention may be employed with respect to any other method orcomposition described herein. Thus, an embodiment pertaining to onemethod or composition may be applied to other methods and compositionsof the invention as well.

As used herein the terms “encode” or “encoding” with reference to anucleic acid are used to make the invention readily understandable bythe skilled artisan however these terms may be used interchangeably with“comprise” or “comprising” respectively.

As used herein the specification, “a” or “an” may mean one or more. Asused herein in the claim(s), when used in conjunction with the word“comprising”, the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: Overview of T-cell reprogramming process, beginning withactivated T-cells and resulting in iPSC colonies with hESC-likemorphology. T-cell and iPSC colony images were acquired on an OlympusIX71 microscope with 10× and 20× objectives, respectively.

FIGS. 2A-2C: Derivation and characterization of induced pluripotent stemcells from human T-cells. (FIG. 2A) Flow cytometric analysis of inputcell source CD3 surface expression. (i) CD3 surface expression on day −3non-activated PBMCs and day 0 activated T-cells from the PBMC populationin a representative donor. (ii) CD3 expression gated on the transduced(GFP⁺) cell population 72 hours post-transduction in a representativedonor to demonstrate preferential transduction of CD3⁺ cells. (iii)Histogram representation of the above metrics (i-ii) in an average of 10donor Vacutainer-derived samples. (FIG. 2B) Flow cytometric analysis ofhESC pluripotency markers OCT4, Tra-1-81, SSEA-3 and SSEA-4 inrepresentative leukapheresis (“TiPS L-2”) and Vacutainer© (“TiPS V-1”)derived TiPS lines. (FIG. 2C) T-cell receptor (TCR) β chainrearrangement analysis using multiplexed PCR primers targeted toconserved regions within the V-J region of the TCR β locus. Polyclonalstarting T-cell populations are represented by a bell-shaped curve ofamplicon peaks within the valid fragment size range on theelectropherogram. Fibroblast (non-T-cell) iPS cells (“Fib-iPS”) lackgermline rearrangement at the TCR β locus and serve as a negativecontrol. The clonally derived TiPS lines (representative data from twoleukapheresis lines and one Vacutainer© line, “TiPS L-1”, “TiPS L-2” and“TiPS V-2”, respectively) show one distinct peak of defined size. DNAfragment analysis was performed on an ABI 3730 DNA analyzer.

FIGS. 3A-3D: Characterization of induced pluripotent stem cells fromhuman T-cells. (FIG. 3A) RT-PCR analysis of representative leukapheresis(“TiPS L-1 and L-2”) and Vacutainer© (“TiPS V-2”) derived TiPS celllines for expression of hES cell-marker genes DNMT38, LEFTB, NODAL,REX1, ESG1, TERT, GDF3, and UTF1. GAPDH was used as positive loadingcontrol for each sample. (FIG. 3B) PCR analysis of genomic DNA confirmsintegration of the transgenes. Forward primers for the reprogramminggene (“RG”) of interest and reverse primers for the IRES were utilized.OCT4 forward and reverse primers were used as the PCR reaction positivecontrol, as shown in vector map. (FIG. 3C) RT-PCR analysis of TiPS celllines shows silencing of the exogenous transgenes, with GAPDH aspositive control for each sample. hESC line H1 and a fibroblast derivediPSC line (Fib-iPS) served as positive cell controls, and activateddonor T-cells served as a negative cell control. (FIG. 3D) TiPS clonesexpressed human embryonic stem cell-specific pluripotency markers asshown by flow cytometry analysis.

FIGS. 4A-4E. in vivo and in vitro differentiation potential of TiPS celllines. (FIG. 4A) Teratoma formation shows in vivo differentiationpotential. TiPS cells injected into SCID/beige mice formed teratomas at5 to 12 weeks. Hematoxylin and eosin staining shows tissues consistentwith derivation from the three primary germ layers including simpleepithelium with goblet cells indicating gastrointestinal or respiratorytissue (endoderm), cartilage (mesoderm) and retinal pigmented epithelium(ectoderm). Representative images from TiPS L-2 cell line were acquiredusing an Olympus IX71 microscope using a 40× objective. (FIG. 4B) Invitro differentiation into neurons. TiPS L-2 cells were induced intoneuronal differentiation as aggregates then stained for neuronal markerbeta III-tubulin with an Alexa Fluor 594 secondary antibody; cell nucleiwere counterstained with Hoechst stain. Images were acquired using a 20×objective. Contrast was adjusted and images were merged using Image Jsoftware. (FIG. 4C) Cardiac induction of TiPS cells via cell aggregatemethod. Cell aggregates contain beating cardiac troponin T(cTNT)-positive cardiomyocytes at day 14 to 15. Flow data fromrepresentative samples is shown. Images were acquired using a 10×objective. (FIG. 4D) In vitro differentiation into hematopoieticprogenitor cells. Hematopoietic progenitor cells (HPCs) generated via aserum-free embryoid body (EB) differentiation protocol for 12 days intwo TiPS lines compared to an hESC line (H1) and a fibroblast derivediPSC line (Fib-iPS). HPCs were quantified via flow cytometry bydissociating the EBs into single cells and staining withfluorochrome-conjugated monoclonal antibodies to CD34, CD45, CD43, CD31,CD41 and CD235a. (FIG. 4E) Hematopoietic clonogenic (CFU) assays wereperformed by placing EB differentiated and individualized cells intoserum-free MethoCult media containing cytokines (SCF, G-CSF, GM-CSF,IL-3, IL-6, and EPO). Colonies were scored after 14 days of incubationaccording to morphologic criteria as erythroid (CFU-E/BFU-E), macrophage(CFU-M, data not shown), granulocyte (CFU-G, data not shown),granulocyte-macrophage (CFU-GM), andgranulocyte-erythroid-macrophage-megakaryocyte (CFU-GEMM). Total CFUcount is also denoted (CFU). Images were acquired using an Olympus CKX41microscope with a 2× objective.

FIG. 5. iPSC Clone Tracking. Genomic DNA was isolated from teratomasamples and compared with their parent cell lines for TCR β chainrearrangements. Representative data is shown from cell line TiPS V-1.The derivative teratoma harbors the clonal rearrangement of the parentcell line. PCR analysis was conducted using multiplexed primers targetedto conserved regions within the V-J region of the TCR β locus. DNAfragment analysis was performed on an ABI 3730 DNA analyzer. Background≦1000 RFU.

FIGS. 6A-6B. Reprogramming T cells in 96-cell format. FIG. 6A. Donor A′T cells are infected with bicistronic vectors SO (Sox2 and Oct4) and CK(c-Myc and Klf4) and plated on MEFs. Live cell anti-Tra1-60 labeling wasconducted to detect iPS cell colonies. FIG. 6B. Donor A′ T cells areinfected with bicistronic vectors SO (Sox2 and Oct4) and NL (Nanog andLin28) and plated on MEFs. Live cell anti-Tra1-60 labeling was conductedto detect iPS cell colonies. Input cell number was shown as “Input #” toindicate the number of T cells in the starting material.

FIG. 7. DNA fingerprinting. Short Tandem Repeat (STR) analysis showsTiPS cell lines are identical to parent activated T-cells for all 15allelic polymorphisms detected across the 8 STR loci analyzed.Representative data from two TiPS lines (TiPS L-1 and TiPS L-2) isshown.

FIG. 8. Alkaline phosphatase (AP) staining. TiPS lines TiPS L-1 and TiPSL-2 are AP positive. Images were acquired on an HP Scanjet G3110computer scanner.

FIG. 9. TiPS cell lines display normal karyotype. TiPS cell lines “TiPSL-1” and “TiPS L-2” were grown for 6 passages on MEFs, and lines “TiPSV-1” and “TiPS V-2” were grown on Matrigel for 8 of 18 total passagesand 30 of 34 total passages, respectively. Cells were subjected to Gbanding analysis and no clonal abnormalities were detected.

FIG. 10. Vector map of the MMLV retroviral construct used forreprogramming experiments. “RG” denotes reprogramming gene.

FIGS. 11A-11C. Vector maps of the bicistronic MMLV retroviral constructsused for reprogramming experiments with improved reprogramming. FIG.11A. Vector map of MMLV-Oct4-IRES-Sox2 (abbreviated as “Oct4-Sox2”).FIG. 11B. Vector map of MMLV-cMyc-IRES-Klf4 (abbreviated as“cMyc-Klf4”). FIG. 11C. Vector map of MMLV-Nanog-IRES-Lin28 (abbreviatedas “Nanog-Lin28”).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. Introduction

In vitro reprogramming of somatic cells to an undifferentiatedpluripotent state by viral transfer of defined factors such as SOX2,OCT4, NANOG and LIN28 or SOX2, OCT4, c-Myc, and KLF4 (Yu et al., 2007;Takahashi et al., 2007b) has opened the way for the generation ofpatient-specific human iPSCs using multiple cell types (Loh et al.,2009; Aasen et al., 2008) This premise has been further advanced byderivation of iPSCs via transient expression of genes or by usingprotein transduction of appropriate transcription factors (Yu et al.,2009; Zhou et al., 2009). To date, the majority of iPSC research inhumans has focused on fibroblasts as a cell source. While fibroblastsoffer certain advantages as a starting material due to their commercialavailability and ease of gene delivery, they are suboptimal forlarge-scale clinical derivation of iPSC lines due to the need forinvasive skin biopsies and the difficulty of establishing stable linesfrom primary tissue. Non-mobilized peripheral blood is perhaps the idealcell source for reprogramming due to the ease of obtaining patientsamples (Maherali and Hochedlinger, 2008). Additionally, large numbersof frozen blood samples, from living and deceased donors, are stored inbiorepositories worldwide (Kleeberger et al., 1999).

The instant invention overcomes several major problems with currentreprogramming technologies by generating induced pluripotent stem cellsfrom T cells and/or hematopoietic precursor cells. As discovered by thepresent invention, more abundant and tractable blood cell source thederivation of iPSCs from T lymphocytes could be obtained from theequivalent of 1 ml whole blood. These T-cell derived iPSCs (“TiPS”)share essential characteristics with hESCs as well as fibroblast-derivediPSC lines. Additionally, they retain their characteristic T-cellreceptor (TCR) gene rearrangements, a property which could be exploited,for example, as a genetic tracking marker or in re-differentiationexperiments to study human T-cell development.

Prior to the present invention, the inventors had significantuncertainties about the likelihood that reprogramming T cells orhematopoietic progenitor cells would be successful for several reasons.First, it was uncertain that whether T cells and/or hematopoieticprecursor cells in blood samples would be present in sufficientquantities for reprogramming. Second, the possible effect of gene lossfrom V(D)J recombination of T-cell receptor genes in reprogramming hadnot been studied. Third, most of the cell types that have beenreprogrammed so far are adherent cell types. T cells are non-adherentand are cultured in suspension. It had not been clear until thisinvention that T cells undergone reprogramming could make the transitionto an adherent culture condition suitable for adherent iPS cells. Thus,methods of the present invention have been the first to enablegeneration of iPS cells from T cells or hematopoietic precursor cells.The T cells can be easily obtained from various sources, such as a smallvolume of blood sample. Similarly, hematopoietic precursor cells, suchas (CD34⁺/CD43⁺/CD45⁺/CD38⁻) or (CD34⁻, CD133⁺/CD38⁻) hematopoieticprecursor cells, may be enriched from a peripheral blood sample.

A particular advantage of the present invention lies in rearranged andreduced V, D, J gene segments of T-cell receptors which may be retainedin reprogrammed progeny cells. This serves as a specific characteristicor “bar code” of different clonal populations of T cell-derived iPScells, and also help differentiates those iPS cells from pluripotentstem cells which have not undergone V(D)J recombination. In addition,the difference in adherent property between T cells and iPS cells makean advantage in automatic separation. Similarly, differences in adherentproperties between hematopoietic precursor cells and iPS cells may beutilized for separation. By transferring reprogrammed T cells orhematopoietic progenitor cells to a culture condition suitable foradherence, such as placing irradiated mouse embryonic fibroblasts (MEFs)on the bottom of the culture vessel for T cells, iPS cells which arederived from the T cells or hematopoietic progenitor cells could adhereto the bottom while the T cells and/or hematopoietic progenitor cellsremain in suspension. Further embodiments and advantages of theinvention are described below.

II. Definitions

“Reprogramming” is a process that confers on a cell a measurablyincreased capacity to form progeny of at least one new cell type, eitherin culture or in vivo, than it would have under the same conditionswithout reprogramming. More specifically, reprogramming is a processthat confers on a somatic cell a pluripotent potential. This means thatafter sufficient proliferation, a measurable proportion of progenyhaving phenotypic characteristics of the new cell type if essentially nosuch progeny could form before reprogramming; otherwise, the proportionhaving characteristics of the new cell type is measurably more thanbefore reprogramming. Under certain conditions, the proportion ofprogeny with characteristics of the new cell type may be at least about1%, 5%, 25% or more in the in order of increasing preference.

An “activator” of a T cell or a condition that will activate a T cellrefers to a stimulus that activates T cells and include antigens, whichmay be presented on antigen presenting cells or on other surfaces;polyclonal activators, which bind to many T cell receptor (TCR)complexes regardless of specificity, and include lectins, e.g.,concanavalin-A (Con-A) and phytohaemagglutinin (PHA) and agents such asantibodies that bind specifically to invariant framework epitopes on TCRor CD3 proteins; and superantigens, which stimulate a significant numberof T cells, and include, e.g., enterotoxins, such as Staphylococcalenterotoxins.

The terms “T lymphocyte” and “T cell” are used interchangeably, andrefer to a cell that expresses a T cell antigen receptor (TCR) capableof recognizing antigen when displayed on the surface of antigenpresenting cells or matrix together with one or more MHC molecules or,one or more non-classical MHC molecules.

“CD4⁺ T cells” refers to a subset of T cells that express CD4 on theirsurface and are associated with cell-mediated immune response. They arecharacterized by the secretion profiles following stimulation, which mayinclude secretion of cytokines such as IFN-gamma, TNF-alpha, IL-2, IL-4and IL-10. “CD4” are 55-kD glycoproteins originally defined asdifferentiation antigens on T-lymphocytes, but also found on other cellsincluding monocytes/macrophages. CD4 antigens are members of theimmunoglobulin supergene family and are implicated as associativerecognition elements in MHC (major histocompatibilit+y complex) classII-restricted immune responses. On T-lymphocytes they define thehelper/inducer subset.

“CD8⁺ T cells” refers to a subset of T cells which express CD8 on theirsurface, are MHC class I-restricted, and function as cytotoxic T cells.“CD8” molecules are differentiation antigens found on thymocytes and oncytotoxic and suppressor T-lymphocytes. CD8 antigens are members of theimmunoglobulin supergene family and are associative recognition elementsin major histocompatibility complex class I-restricted interactions.

“Hematopoietic progenitor cells” or “hematopoietic precursor cells”refers to cells which are committed to a hematopoietic lineage but arecapable of further hematopoietic differentiation and includehematopoietic stem cells, multipotential hematopoietic stem cells(hematoblasts), myeloid progenitors, megakaryocyte progenitors,erythrocyte progenitors, and lymphoid progenitors. Hematopoietic stemcells (HSCs) are multipotent stem cells that give rise to all the bloodcell types including myeloid (monocytes and macrophages, neutrophils,basophils, eosinophils, erythrocytes, megakaryocytes/platelets,dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells).The hematopoietic progenitor cells may express CD34. The hematopoieticprogenitor cells may co-express CD133 and be negative for CD38expression. In certain embodiments, certain human hematopoietic may notexpress CD34, but these cells may nonetheless be converted into iPScells via the methods disclosed herein. Hematopoietic precursor cellsinclude CD34⁺/CD45⁺ hematopoietic precursor cells and CD34⁺/CD45⁺/CD43⁺hematopoietic precursor cells. The CD34⁺/CD43⁺/CD45⁺ hematopoieticprecursor cells may be highly enriched for myeloid progenitors. Variouslineages of hematopoietic cells, such as CD34⁺/CD43⁺/CD45⁺ hematopoieticprecursor cells, may be converted to iPS cells via the methods disclosedherein. Hematopoietic cells also include various subsets of primitivehematopoietic cells including: CD34⁻/CD133⁺/CD38⁻ (primitivehematopoietic precursor cells), CD43(+)CD235a(+)CD41a(+/−)(erythro-megakaryopoietic), lin(−)CD34(+)CD43(+)CD45(−) (multipotent),and lin(+)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells,CD133+/ALDH+(aldehydehehydrogenase) (e.g., Hess et al. 2004; Christ etal., 2007). It is anticipated that any of these primitive hematopoieticcell types or hematopoietic precursor cells may be converted into iPScells as described herein.

A “vector” or “construct” (sometimes referred to as gene delivery orgene transfer “vehicle”) refers to a macromolecule or complex ofmolecules comprising a polynucleotide to be delivered to a host cell,either in vitro or in vivo. A vector can be a linear or a circularmolecule.

A “plasmid”, a common type of a vector, is an extra-chromosomal DNAmolecule separate from the chromosomal DNA which is capable ofreplicating independently of the chromosomal DNA. In certain cases, itis circular and double-stranded.

By “expression construct” or “expression cassette” is meant a nucleicacid molecule that is capable of directing transcription. An expressionconstruct includes, at the least, a promoter or a structure functionallyequivalent to a promoter. Additional elements, such as an enhancer,and/or a transcription termination signal, may also be included.

The term “exogenous,” when used in relation to a protein, gene, nucleicacid, or polynucleotide in a cell or organism refers to a protein, gene,nucleic acid, or polynucleotide which has been introduced into the cellor organism by artificial or natural means, or in relation a cell refersto a cell which was isolated and subsequently introduced to other cellsor to an organism by artificial or natural means. An exogenous nucleicacid may be from a different organism or cell, or it may be one or moreadditional copies of a nucleic acid which occurs naturally within theorganism or cell. An exogenous cell may be from a different organism, orit may be from the same organism. By way of a non-limiting example, anexogenous nucleic acid is in a chromosomal location different from thatof natural cells, or is otherwise flanked by a different nucleic acidsequence than that found in nature.

The term “corresponds to” is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term “complementary to” is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence “TATAC” corresponds to a reference sequence “TATAC”and is complementary to a reference sequence “GTATA”.

A “gene,” “polynucleotide,” “coding region,” “sequence,” “segment,”“fragment,” or “transgene” which “encodes” a particular protein, is anucleic acid molecule which is transcribed and optionally alsotranslated into a gene product, e.g., a polypeptide, in vitro or in vivowhen placed under the control of appropriate regulatory sequences. Thecoding region may be present in either a cDNA, genomic DNA, or RNA form.When present in a DNA form, the nucleic acid molecule may besingle-stranded (i.e., the sense strand) or double-stranded. Theboundaries of a coding region are determined by a start codon at the 5′(amino) terminus and a translation stop codon at the 3′ (carboxy)terminus. A gene can include, but is not limited to, cDNA fromprokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryoticor eukaryotic DNA, and synthetic DNA sequences. A transcriptiontermination sequence will usually be located 3′ to the gene sequence.

The term “cell” is herein used in its broadest sense in the art andrefers to a living body which is a structural unit of tissue of amulticellular organism, is surrounded by a membrane structure whichisolates it from the outside, has the capability of self replicating,and has genetic information and a mechanism for expressing it. Cellsused herein may be naturally-occurring cells or artificially modifiedcells (e.g., fusion cells, genetically modified cells, etc.).

As used herein, the term “stem cell” refers to a cell capable of selfreplication and pluripotency. Typically, stem cells can regenerate aninjured tissue. Stem cells herein may be, but are not limited to,embryonic stem (ES) cells or tissue stem cells (also calledtissue-specific stem cell, or somatic stem cell). Any artificiallyproduced cell which can have the above-described abilities (e.g., fusioncells, reprogrammed cells, or the like used herein) may be a stem cell.

“Embryonic stem (ES) cells” are pluripotent stem cells derived fromearly embryos. An ES cell was first established in 1981, which has alsobeen applied to production of knockout mice since 1989. In 1998, a humanES cell was established, which is currently becoming available forregenerative medicine.

Unlike ES cells, tissue stem cells have a limited differentiationpotential. Tissue stem cells are present at particular locations intissues and have an undifferentiated intracellular structure. Therefore,the pluripotency of tissue stem cells is typically low. Tissue stemcells have a higher nucleus/cytoplasm ratio and have few intracellularorganelles. Most tissue stem cells have low pluripotency, a long cellcycle, and proliferative ability beyond the life of the individual.Tissue stem cells are separated into categories, based on the sites fromwhich the cells are derived, such as the dermal system, the digestivesystem, the bone marrow system, the nervous system, and the like. Tissuestem cells in the dermal system include epidermal stem cells, hairfollicle stem cells, and the like. Tissue stem cells in the digestivesystem include pancreatic (common) stem cells, liver stem cells, and thelike. Tissue stem cells in the bone marrow system include hematopoieticstem cells, mesenchymal stem cells, and the like. Tissue stem cells inthe nervous system include neural stem cells, retinal stem cells, andthe like.

“Induced pluripotent stem cells,” commonly abbreviated as iPS cells oriPSCs, refer to a type of pluripotent stem cell artificially preparedfrom a non-pluripotent cell, typically an adult somatic cell, orterminally differentiated cell, such as fibroblast, a hematopoieticcell, a myocyte, a neuron, an epidermal cell, or the like, byintroducing certain factors, referred to as reprogramming factors.

“Pluripotency” refers to a stem cell that has the potential todifferentiate into all cells constituting one or more tissues or organs,or particularly, any of the three germ layers: endoderm (interiorstomach lining, gastrointestinal tract, the lungs), mesoderm (muscle,bone, blood, urogenital), or ectoderm (epidermal tissues and nervoussystem). “Pluripotent stem cells” used herein refer to cells that candifferentiate into cells derived from any of the three germ layers, forexample, direct descendants of totipotent cells or induced pluripotentcells.

By “operably linked” with reference to nucleic acid molecules is meantthat two or more nucleic acid molecules (e.g., a nucleic acid moleculeto be transcribed, a promoter, and an enhancer element) are connected insuch a way as to permit transcription of the nucleic acid molecule.“Operably linked” with reference to peptide and/or polypeptide moleculesis meant that two or more peptide and/or polypeptide molecules areconnected in such a way as to yield a single polypeptide chain, i.e., afusion polypeptide, having at least one property of each peptide and/orpolypeptide component of the fusion. The fusion polypeptide isparticularly chimeric, i.e., composed of heterologous molecules.

“Homology” refers to the percent of identity between two polynucleotidesor two polypeptides. The correspondence between one sequence and toanother can be determined by techniques known in the art. For example,homology can be determined by a direct comparison of the sequenceinformation between two polypeptide molecules by aligning the sequenceinformation and using readily available computer programs.Alternatively, homology can be determined by hybridization ofpolynucleotides under conditions which form stable duplexes betweenhomologous regions, followed by digestion with single strand-specificnuclease(s), and size determination of the digested fragments. Two DNA,or two polypeptide, sequences are “substantially homologous” to eachother when at least about 80%, particularly at least about 90%, and mostparticularly at least about 95% of the nucleotides, or amino acids,respectively match over a defined length of the molecules, as determinedusing the methods above.

III. General Background for Stem Cells

In certain embodiments of the invention, there are disclosed methods ofreprogramming somatic cells, especially T cell, by introducingreprogramming factors into somatic cells. The progeny of these cellscould be identical to embryonic stem cells in various aspects asdescribed below, but essentially free of exogenous genetic elements.Understanding of embryonic stem cell characteristics could help selectinduced pluripotent stem cells. Reprogramming factors known from stemcell reprogramming studies could be used for these novel methods. It isfurther contemplated that these induced pluripotent stem cells could bepotentially used to replace embryonic stem cells for therapeutics andresearch applications due to the ethics hurdle to use the latter.

A. Stem Cells

Stem cells are cells found in most, if not all, multi-cellularorganisms. They are characterized by the ability to renew themselvesthrough mitotic cell division and differentiating into a diverse rangeof specialized cell types. The two broad types of mammalian stem cellsare: embryonic stem cells that are found in blastocysts, and adult stemcells that are found in adult tissues. In a developing embryo, stemcells can differentiate into all of the specialized embryonic tissues.In adult organisms, stem cells and progenitor cells act as a repairsystem for the body, replenishing specialized cells, but also maintainthe normal turnover of regenerative organs, such as blood, skin orintestinal tissues.

As stem cells can be grown and transformed into specialized cells withcharacteristics consistent with cells of various tissues such as musclesor nerves through cell culture, their use in medical therapies has beenproposed. In particular, embryonic cell lines, autologous embryonic stemcells generated through therapeutic cloning, and highly plastic adultstem cells from the umbilical cord blood or bone marrow are touted aspromising candidates. Most recently, the reprogramming of adult cellsinto induced pluripotent stem cells has enormous potential for replacingembryonic stem cells.

B. Embryonic Stem Cells

Embryonic stem cell lines (ES cell lines) are cultures of cells derivedfrom the epiblast tissue of the inner cell mass (ICM) of a blastocyst orearlier morula stage embryos. A blastocyst is an early stageembryo—approximately four to five days old in humans and consisting of50-150 cells. ES cells are pluripotent and give rise during developmentto all derivatives of the three primary germ layers: ectoderm, endodermand mesoderm. In other words, they can develop into each of the morethan 200 cell types of the adult body when given sufficient andnecessary stimulation for a specific cell type. They do not contributeto the extra-embryonic membranes or the placenta.

Nearly all research to date has taken place using mouse embryonic stemcells (mES) or human embryonic stem cells (hES). Both have the essentialstem cell characteristics, yet they require very different environmentsin order to maintain an undifferentiated state. Mouse ES cells may begrown on a layer of gelatin and require the presence of LeukemiaInhibitory Factor (LIF). Human ES cells could be grown on a feeder layerof mouse embryonic fibroblasts (MEFs) and often require the presence ofbasic Fibroblast Growth Factor (bFGF or FGF-2). Without optimal cultureconditions or genetic manipulation (Chambers et al., 2003), embryonicstem cells will rapidly differentiate.

A human embryonic stem cell may be also defined by the presence ofseveral transcription factors and cell surface proteins. Thetranscription factors Oct4, Nanog, and Sox2 form the core regulatorynetwork that ensures the suppression of genes that lead todifferentiation and the maintenance of pluripotency (Boyer et al.,2005). The cell surface antigens most commonly used to identify hEScells include the glycolipids SSEA3 and SSEA4 and the keratan sulfateantigens Tra-1-60 and Tra-1-81.

After twenty years of research, there are no approved treatments orhuman trials using embryonic stem cells. ES cells, being pluripotentcells, require specific signals for correct differentiation—if injecteddirectly into the body, ES cells will differentiate into many differenttypes of cells, causing a teratoma. Differentiating ES cells into usablecells while avoiding transplant rejection are just a few of the hurdlesthat embryonic stem cell researchers still face. Many nations currentlyhave moratoria on either ES cell research or the production of new EScell lines. Because of their combined abilities of unlimited expansionand pluripotency, embryonic stem cells remain a theoretically potentialsource for regenerative medicine and tissue replacement after injury ordisease. However, one way to circumvent these issues is to inducepluripotent status in somatic cells by direct reprogramming.

IV. Reprogramming Factors

The generation of iPS cells is crucial on the reprogramming factors usedfor the induction. The following factors or combination thereof could beused in the methods disclosed in the present invention. In certainaspects, nucleic acids encoding Sox and Oct (particularly Oct3/4) willbe included into the reprogramming vector. For example, one or morereprogramming vectors may comprise expression cassettes encoding Sox2,Oct4, Nanog and optionally Lin28, or expression cassettes encoding Sox2,Oct4, Klf4 and optionally c-Myc, or expression cassettes encoding Sox2,Oct4, and optionally Esrrb, or expression cassettes encoding Sox2, Oct4,Nanog, Lin28, Klf4, c-Myc, and optionally SV40 Large T antigen. Nucleicacids encoding these reprogramming factors may be comprised in the sameexpression cassette, different expression cassettes, the samereprogramming vector, or different reprogramming vectors.

Oct4 and certain members of the Sox gene family (Sox1, Sox2, Sox3, andSox15) have been identified as crucial transcriptional regulatorsinvolved in the induction process whose absence makes inductionimpossible. Additional genes, however, including certain members of theKlf family (Klf1, Klf2, Klf4, and Klf5), the Myc family (c-Myc, L-Myc,and N-Myc), Nanog, and Lin28, have been identified to increase theinduction efficiency.

Oct4 (Pou5f1) is one of the family of octamer (“Oct”) transcriptionfactors, and plays a crucial role in maintaining pluripotency. Theabsence of Oct4 in Oct4⁺ cells, such as blastomeres and embryonic stemcells, leads to spontaneous trophoblast differentiation, and presence ofOct4 thus gives rise to the pluripotency and differentiation potentialof embryonic stem cells. Various other genes in the “Oct” family,including Oct4's close relatives, Oct1 and Oct6, fail to elicitinduction, thus demonstrating the exclusiveness of Oct-4 to theinduction process.

The Sox family of genes is associated with maintaining pluripotencysimilar to Oct4, although it is associated with multipotent andunipotent stem cells in contrast with Oct4, which is exclusivelyexpressed in pluripotent stem cells. While Sox2 was the initial geneused for reprogramming induction, other genes in the Sox family havebeen found to work as well in the induction process. Sox1 yields iPScells with a similar efficiency as Sox2, and genes Sox3, Sox15, andSox18 also generate iPS cells, although with decreased efficiency.

In embryonic stem cells, Nanog, along with Oct4 and Sox2, is necessaryin promoting pluripotency. Therefore, it was surprising when Yamanaka etal. reported that Nanog was unnecessary for induction although Thomsonet al. has reported it is possible to generate iPS cells with Nanog asone of the factors.

Lin28 is an mRNA binding protein expressed in embryonic stem cells andembryonic carcinoma cells associated with differentiation andproliferation. Thomson et al. demonstrated it is a factor in iPSgeneration, although it is unnecessary.

Klf4 of the Klf family of genes was initially identified by Yamanaka etal. and confirmed by Jaenisch et al. as a factor for the generation ofmouse iPS cells and was demonstrated by Yamanaka et al. as a factor forgeneration of human iPS cells. However, Thompson et al. reported thatKlf4 was unnecessary for generation of human iPS cells and in factfailed to generate human iPS cells. Klf2 and Klf4 were found to befactors capable of generating iPS cells, and related genes Klf 1 andKlf5 did as well, although with reduced efficiency.

The Myc family of genes are proto-oncogenes implicated in cancer.Yamanaka et al. and Jaenisch et al. demonstrated that c-Myc is a factorimplicated in the generation of mouse iPS cells and Yamanaka et al.demonstrated it was a factor implicated in the generation of human iPScells. However, Thomson et al. and Yamanaka et al. reported that c-Mycwas unnecessary for generation of human iPS cells. Usage of the “Myc”family of genes in induction of iPS cells is troubling for theeventuality of iPS cells as clinical therapies, as 25% of micetransplanted with c-Myc-induced iPS cells developed lethal teratomas.N-Myc and L-Myc have been identified to induce in the stead of c-mycwith similar efficiency. SV40 large antigen may be used to reduce orprevent the cytotoxcity which may occur when c-Myc is expressed.

The reprogramming proteins used in the present invention can besubstituted by protein homologs with about the same reprogrammingfunctions. Nucleic acids encoding those homologs could also be used forreprogramming. Conservative amino acid substitutions are preferred—thatis, for example, aspartic-glutamic as polar acidic amino acids;lysine/arginine/histidine as polar basic amino acids;leucine/isoleucine/methionine/valine/alanine/glycine/proline asnon-polar or hydrophobic amino acids; serine/threonine as polar oruncharged hydrophilic amino acids. Conservative amino acid substitutionalso includes groupings based on side chains. For example, a group ofamino acids having aliphatic side chains is glycine, alanine, valine,leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine. Forexample, it is reasonable to expect that replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid will not have a major effect on the properties of theresulting polypeptide. Whether an amino acid change results in afunctional polypeptide can readily be determined by assaying thespecific activity of the polypeptide.

V. Reprogramming of T Cells and/or Hematopoietic Precursor Cells

To provide iPS cells from alternative sources in addition to dermalfibroblasts commonly used in the current art, methods for reprogramminga cell population comprising T cells are provided. In certainembodiments, T cells are activated and expanded to provide a significantnumber of T cells for reprogramming.

A. T Cells

The term “T cell” refers to T lymphocytes as defined in the art and isintended to include thymocytes, immature T lymphocytes, mature Tlymphocytes, resting T lymphocytes, or activated T lymphocytes. The Tcells can be CD4⁺ T cells, CD8⁺ T cells, CD4⁺CD8⁺ T cells, or CD4⁻CD8⁻cells. The T cells can also be T helper cells, such as T helper 1 (TH1),or T helper 2 (TH2) cells, or TH17 cells, as well as cytotoxic T cells,regulatory T cells, natural killer T cells, naïve T cells, memory Tcells, or gamma delta T cells (Wilson et al., 2009; Wynn, 2005; Ladi etal., 2006). T cells that differ from each other by at least one marker,such as CD4, are referred to herein as “subsets” of T cells.

Helper T cells (effector T cells or Th cells) are the “middlemen” of theadaptive immune system. Once activated, they divide rapidly and secretesmall proteins called cytokines that regulate or assist in the immuneresponse. Depending on the size, cytokine signals received, these cellsdifferentiate into TH1, TH2, TH3, TH17, THF, or one of other subsets,which secrete different cytokines. CD4⁺ cells are associated with MHCclass II.

Cytotoxic T cells (TC cells, or CTLs) destroy virally infected cells andtumor cells, and are also implicated in transplant rejection. Thesecells are also known as CD8⁺ T cells (associated with MHC class I),since they express the CD8 glycoprotein at their surface. Through SLOBinteraction with T regulatory CD4⁺CD25⁺FoxP3⁺ cells, these cells can beinactivated to a anergic state, which prevent autoimmune diseases suchas experimental autoimmune encephalomyelitis.

Memory T cells are a subset of antigen-specific T cells that persistlong-term after an infection has resolved. They quickly expand to largenumbers of effector T cells upon re-exposure to their cognate antigen,thus providing the immune system with “memory” against past infections.Memory T cells comprise two subtypes: central memory T cells (TCM cells)and effector memory T cells (TEM cells). Memory cells may be either CD4⁺or CD8⁺.

Regulatory T cells (Treg cells), formerly known as suppressor T cells,are crucial for the maintenance of immunological tolerance. Theyresemble the conventional alpha beta TCR expressing CD4 positive cells.They can be further characterized by the co expression of CD25 and Foxp3proteins. Their major role is to shut down T cell-mediated immunitytoward the end of an immune reaction and to suppress auto-reactive Tcells that escaped the process of negative selection in the thymus. Twomajor classes of CD4⁺ regulatory T cells have been described, includingthe naturally occurring Treg cells and the adaptive Treg cells.Naturally occurring Treg cells (also known as CD4⁺CD25⁺ FoxP3⁺ Tregcells) arise in the thymus, whereas the adaptive Treg cells (also knownas Tr1 cells or Th3 cells) may originate during a normal immuneresponse. Naturally occurring Treg cells can be distinguished from otherT cells by the presence of an intracellular molecule called FoxP3.Mutations of the FOXP3 gene can prevent regulatory T cell development,causing the fatal autoimmune disease IPEX.

Natural killer T cells (NK T cells) are a heterogeneous T cellpopulation characterized by the co-expression of αβ or γδ TCRs andvarious NK receptors, including CD16, CD56, CD161, CD94, CD158a andCD158b NK T cells have the ability to rapidly secrete large amounts ofcytokines following activation. NK T cell clones secrete type 1, type 2or both types of cytokines, which could influence the differentiation ofTh0 cells towards Th1 or Th2 cells. NK T cells were described as cellsthat express an invariant TCR Valpha14 in mouse and Valpha24 in humans.Recently NK T cells expressing diverse TCRs have been also recognizedCD3⁺CD56⁺ cells represent one of the NK T cell populations NK T cellscan be CD4⁺CD8⁺, CD4⁻CD8⁻, CD4⁻CD8⁺, or CD4⁺CD8⁻.

γδ T cells (gamma delta T cells) represent a small subset of T cellsthat possess a distinct T cell receptor (TCR) on their surface. Amajority of T cells have a TCR composed of two glycoprotein chainscalled α- and β-TCR chains. However, in γδ T cells, the TCR is made upof one γ-chain and one δ-chain. This group of T cells is much lesscommon (5% of total T cells) than the αβ T cells, but are found at theirhighest abundance in the gut mucosa, within a population of lymphocytesknown as intraepithelial lymphocytes (IELs). The antigenic moleculesthat activate γδ T cells are still widely unknown. However, γδ T cellsare not MHC restricted and seem to be able to recognize whole proteinsrather than requiring peptides to be presented by MHC molecules onantigen presenting cells. Some recognize MHC class IB molecules though.Human Vγ9/Vδ2 T cells, which constitute the major γδ T cell populationin peripheral blood, are unique in that they specifically and rapidlyrespond to a small non-peptidic microbial metabolite, HMB-PP, anisopentenyl pyrophosphate precursor. Estimates of the percentages of Tcells that may be found in peripheral blood from healthy donors are asfollows: CD3⁺=70.78%±4.71; CD3⁺CD4=38.97%±5.66; CD3⁺CD8=28.955%±7.43;CD3⁺CD56⁺=5.22%±1.74; CD3⁻CD56⁺=10.305%±4.7; CD3⁺CD45RA=45.00%±7.19;CD3⁺CD45RO⁺=27.21%±7.34.

The T cells can be a purified population of T cells, or alternativelythe T cells can be in a population with cells of a different type, suchas B cells and/or other peripheral blood cells. The T cells can be apurified population of a subset of T cells, such as CD4⁺ T cells, orthey can be a population of T cells comprising different subsets of Tcells. In another embodiment of the invention, the T cells are T cellclones that have been maintained in culture for extended periods oftime. T cell clones can be transformed to different degrees. In aspecific embodiment, the T cells are a T cell clone that proliferatesindefinitely in culture.

In a preferred embodiment of the invention, the T cells are primary Tcells. The term “primary T cells” is intended to include T cellsobtained from an individual, as opposed to T cells that have beenmaintained in culture for extended periods of time. Thus, primary Tcells are particularly peripheral blood T cells obtained from a subject.A population of primary T cells can be composed of mostly one subset ofT cells. Alternatively, the population of primary T cells can becomposed of different subsets of T cells.

The T cells can be from previously stored blood samples, from a healthyindividual, or alternatively from an individual affected with acondition. The condition can be an infectious disease, such as acondition resulting from a viral infection, a bacterial infection or aninfection by any other microorganism, or a hyperproliferative disease,such as cancer like melanoma. In a specific embodiment, the T cells arefrom an individual infected with a human immunodeficiency virus (HIV).In yet another embodiment of the invention, the T cells are from asubject suffering from or susceptible to an autoimmune disease or T-cellpathologies. The T cells can be of human origin, murine origin or anyother mammalian species.

B. Hematopoietic Progenitor Cells

Due to the significant medical potential of hematopoietic stem andprogenitor cells, substantial work has been done to try to improvemethods for the differentiation of hematopoietic progenitor cells fromembryonic stem cells. In the human adult, hematopoietic stem cellspresent primarily in bone marrow produce heterogeneous populations ofactively dividing hematopoietic (CD34⁺) progenitor cells thatdifferentiate into all the cells of the blood system. While it isanticipated that CD34⁺ endothelial cells may be converted to iPS cells,in certain embodiments it may be desirable to use hematopoietic cellswhich are not endothelial cells; for example, in some instances it maybe desirable to use hematopoietic progenitor cells or hematopoieticprecursor cells which do not express CD31 or VE-cadherin. Other markers,such as the CD43 and/or CD45 marker, may also be used to help identifyhematopoietic progenitor cells (e.g., Kadaja-Saarepuu et al., 2008;Vodyanik et al., 2006). Hematopoietic cells include various subsets ofprimitive hematopoietic cells including: CD43(+)CD235a(+)CD41a(+/−)(erythro-megakaryopoietic), lin(+)CD34(+)CD43(+)CD45(−) (multipotent),and lin(−)CD34(+)CD43(+)CD45(+) (myeloid-skewed) cells. In an adulthuman, hematopoietic progenitors proliferate and differentiate resultingin the generation of hundreds of billions of mature blood cells daily.Hematopoietic progenitor cells are also present in cord blood. In vitro,human embryonic stem cells may be differentiated into hematopoieticprogenitor cells. Hematopoietic progenitor cells may also be expanded orenriched from a sample of peripheral blood. The hematopoietic cells canbe of human origin, murine origin or any other mammalian species.

C. Sources of Populations of Cells

Hematopoietic stem cells (HSCs) normally reside in the bone marrow butcan be forced into the blood, a process termed mobilization usedclinically to harvest large numbers of HSCs in peripheral blood. Onemobilizing agent of choice is granulocyte colony-stimulating factor(G-CSF).

CD34⁺ hematopoietic stem cells or progenitors that circulate in theperipheral blood can be collected by apheresis techniques either in theunperturbed state, or after mobilization following the externaladministration of hematopoietic growth factors like G-CSF. The number ofthe stem or progenitor cells collected following mobilization is greaterthan that obtained after apheresis in the unperturbed state. In aparticular aspect of the present invention, the source of the cellpopulation is a subject whose cells have not been mobilized byextrinsically applied factors because there is no need to enrichhematopoietic stem cells or progenitor cells.

Populations of cells for use in the methods described herein may bemammalian cells, such as human cells, non-human primate cells, rodentcells (e.g., mouse or rat), bovine cells, ovine cells, porcine cells,equine cells, sheep cell, canine cells, and feline cells or a mixturethereof. Non-human primate cells include rhesus macaque cells. The cellsmay be obtained from an animal, e.g., a human patient, or they may befrom cell lines. If the cells are obtained from an animal, they may beused as such, e.g., as unseparated cells (i.e., a mixed population);they may have been established in culture first, e.g., bytransformation; or they may have been subjected to preliminarypurification methods. For example, a cell population may be manipulatedby positive or negative selection based on expression of cell surfacemarkers; stimulated with one or more antigens in vitro or in vivo;treated with one or more biological modifiers in vitro or in vivo; or acombination of any or all of these. In an illustrative embodiment, acell population is subjected to negative selection for depletion ofnon-T cells and/or particular T cell subsets. Negative selection can beperformed on the basis of cell surface expression of a variety ofmolecules, including B cell markers such as CD19, and CD20; monocytemarker CD14; the NK cell marker CD56. Alternately, a cell population maybe subjected to negative selection for depletion of non-CD34⁺hematopoietic cells and/or particular hematopoietic cell subsets.Negative selection can be performed on the basis of cell surfaceexpression of a variety of molecules, such as a cocktail of antibodies(e.g., CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a)which may be used for separation of other cell types, e.g., via MACS orcolumn separation.

Populations of cells include peripheral blood mononuclear cells (PBMC),whole blood or fractions thereof containing mixed populations, spleencells, bone marrow cells, tumor infiltrating lymphocytes, cells obtainedby leukapheresis, biopsy tissue, lymph nodes, e.g., lymph nodes drainingfrom a tumor. Suitable donors include immunized donors, non-immunized(naive) donors, treated or untreated donors. A “treated” donor is onethat has been exposed to one or more biological modifiers. An“untreated” donor has not been exposed to one or more biologicalmodifiers.

Methods of obtaining populations of cells comprising a T cell are wellknown in the art. For example, peripheral blood mononuclear cells (PBMC)can be obtained as described according to methods known in the art.Examples of such methods are set forth in the Examples and is discussedby Kim et al. (1992); Biswas et al. (1990); Biswas et al. (1991).

Methods of obtaining hematopoietic precursor cells from populations ofcells are also well known in the art. Hematopoietic precursor cells maybe expanded using various cytokines, such as hSCF, hFLT3, and/or IL-3(Akkina et al., 1996), or CD34⁺ cells may be enriched using MACS orFACS. As mentioned above, negative selection techniques may also be usedto enrich CD34⁺ cells.

It is also possible to obtain a cell sample from a subject, and then toenrich it for a desired cell type. For example, PBMCs and/or CD34⁺hematopoietic cells can be isolated from blood as described herein.Counter-flow centrifugation (elutriation) can be used to enrich for Tcells from PBMCs. Cells can also be isolated from other cells using avariety of techniques, such as isolation and/or activation with anantibody binding to an epitope on the cell surface of the desired celltype, for example, some T-cell isolation kits use antibody conjugatedbeads to both activate the cells and then allow column separation withthe same beads. Another method that can be used includes negativeselection using antibodies to cell surface markers to selectively enrichfor a specific cell type without activating the cell by receptorengagement.

Bone marrow cells may be obtained from iliac crest, femora, tibiae,spine, rib or other medullary spaces. Bone marrow may be taken out ofthe patient and isolated through various separations and washingprocedures. A known procedure for isolation of bone marrow cellscomprises the following steps: a) centrifugal separation of bone marrowsuspension in three fractions and collecting the intermediate fraction,or buffycoat; b) the buffycoat fraction from step (a) is centrifuged onemore time in a separation fluid, commonly Ficoll (a trademark ofPharmacia Fine Chemicals AB), and an intermediate fraction whichcontains the bone marrow cells is collected; and c) washing of thecollected fraction from step (b) for recovery of re-transfusable bonemarrow cells.

If one desires to use a population of cells enriched in T cells, suchpopulations of cells can be obtained from a mixed population of cells byleukapheresis and mechanical apheresis using a continuous flow cellseparator. For example, T cells can be isolated from the buffy coat byany known method, including separation over Ficoll-Hypaque™ gradient,separation over a Percoll gradient, or elutriation.

D. T Cell Activation

In certain aspects, T cells are activated by agents that bind to T cellreceptors to trigger a signaling cascade for T cell activation. Forexample, a CD3 antibody may be used. For T cell expansion to asignificant number and a proliferating state for reprogramming, acytokine may also be used, such as IL-2.

Naive T cells can live for many years without dividing. These smallresting cells have condensed chromatin and a scanty cytoplasm andsynthesize little RNA or protein. On activation, they must reenter thecell cycle and divide rapidly to produce the large numbers of progenythat will differentiate into armed effector T cells. Their proliferationand differentiation are driven by a cytokine called interleukin-2(IL-2), which is produced by the activated T cell itself.

The initial encounter with specific antigen in the presence of therequired co-stimulatory signal triggers entry of the T cell into the G₁phase of the cell cycle; at the same time, it also induces the synthesisof IL-2 along with the α chain of the IL-2 receptor. The IL-2 receptorhas three chains: α, β, and γ. Resting T cells express a form of thisreceptor composed of β and γ chains which binds IL-2 with moderateaffinity, allowing resting T cells to respond to very highconcentrations of IL-2. Association of the α chain with the β and γchains creates a receptor with a much higher affinity for IL-2, allowingthe cell to respond to very low concentrations of IL-2. Binding of IL-2to the high-affinity receptor then triggers progression through the restof the cell cycle. T cells activated in this way can divide two to threetimes a day for several days, allowing one cell to give rise to a clonecomposed of thousands of progeny that all bear the same receptor forantigen. IL-2 also promotes the differentiation of these cells intoarmed effector T cells.

Although the specific mechanisms of activation vary slightly betweendifferent types of T cells, the “two-signal model” in CD4⁺ T cells holdstrue for most. Activation of CD4⁺ T cells occurs through the engagementof both the T cell receptor and CD28 on the T cell by the Majorhistocompatibility complex peptide and B7 family members on the APC,respectively. Both are required for production of an effective immuneresponse; in the absence of CD28 co-stimulation, T cell receptorsignalling alone results in anergy. The signalling pathways downstreamfrom both CD28 and the T cell receptor involve many proteins.

The first signal is provided by binding of the T cell receptor to ashort peptide presented by the major histocompatibility complex (MHC) onanother cell. This ensures that only a T cell with a TCR specific tothat peptide is activated. The partner cell is usually a professionalantigen presenting cell (APC), usually a dendritic cell in the case ofnaïve responses, although B cells and macrophages can be important APCs.The peptides presented to CD8⁺ T cells by MHC class I molecules are 8-9amino acids in length; the peptides presented to CD4⁺ cells by MHC classII molecules are longer, as the ends of the binding cleft of the MHCclass II molecule are open.

The second signal comes from co-stimulation, in which surface receptorson the APC are induced by a relatively small number of stimuli, usuallyproducts of pathogens, but sometimes breakdown products of cells, suchas necrotic-bodies or heat-shock proteins. The only co-stimulatoryreceptor expressed constitutively by naïve T cells is CD28, soco-stimulation for these cells comes from the CD80 and CD86 proteins onthe APC. Other receptors are expressed upon activation of the T cell,such as OX40 and ICOS, but these largely depend upon CD28 for theirexpression. The second signal licenses the T cell to respond to anantigen. Without it, the T cell becomes anergic, and it becomes moredifficult for it to activate in future. This mechanism preventsinappropriate responses to self, as self-peptides will not usually bepresented with suitable co-stimulation.

In a certain aspect, both anti-CD3 and anti-CD28 may be used for T cellactivation where co-stimulation is involved. In an alternative aspect,cross-linking of the anti-CD3 may be applied, such as plate boundanti-CD3. If soluble anti-CD3 is used to activate T cells in PBMC, thesoluble anti-CD3 antibody may bind to APCs in the PBMC, which thenpresents the antibody to the T cells. If the soluble anti-CD3 antibodyalone is used in a population of purified T-cells, anergy would resultfor the reasons mentioned above. A certain embodiment of the presentinvention comprises culturing T cells in the presence of the anti-CD3(OKT3) and IL2, which is advantageous and convenient because there is noneed to use costly and cumbersome beads or plate-bound antibody; afteradding OKT3 and IL2, the cellular milieu of PBMCs would help activatethe T cells. The T cells then overcrowd the other cell types in the PBMCculture due to preferential expansion.

The T cell receptor exists as a complex of several proteins. The actualT cell receptor is composed of two separate peptide chains, which areproduced from the independent T cell receptor alpha and beta (TCRα andTCRβ) genes. The other proteins in the complex are the CD3 proteins:CD3εγ and CD3εδ heterodimers and, most important, a CD3ξ homodimer,which has a total of six ITAM motifs. The ITAM motifs on the CD3ξ can bephosphorylated by Lck and in turn recruit ZAP-70. Lck and/or ZAP-70 canalso phosphorylate the tyrosines on many other molecules, not leastCD28, LAT and SLP-76, which allows the aggregation of signallingcomplexes around these proteins.

Phosphorylated LAT recruits SLP-76 to the membrane, where it can thenbring in PLCγ, VAV1, Itk and potentially PI3K. Both PLCγ and PI3K act onPI(4,5)P2 on the inner leaflet of the membrane to create the activeintermediaries diacylglycerol (DAG), inositol-1,4,5-trisphosphate (IP3),and phosphatidylinositol-3,4,5-trisphosphate (PIP3). DAG binds andactivates some PKCs in T cells, e.g., PKCθ, a process important foractivating the transcription factors NF-κB and AP-1. IP3 is releasedfrom the membrane by PLCγ and diffuses rapidly to activate receptors onthe ER, which induce the release of calcium. The released calcium thenactivates calcineurin, and calcineurin activates NFAT, which thentranslocates to the nucleus. NFAT is a transcription factor, whichactivates the transcription of a pleiotropic set of genes, most notable,IL-2, a cytokine that promotes long term proliferation of activated Tcells.

According to the method of the invention, the nucleic acid molecule isintroduced into T cells that are actively proliferating (i.e.,expanding). T cells can be stimulated to expand by contacting the Tcells with a variety of agents, such as a combination of agentsproviding a primary activation signal and a costimulatory signal to Tcells. Agents that can be used to stimulate T cells to expand are wellknown in the art and are described below. T cells that are stimulated toproliferate are characterized by cellular enlargement, clumping, andacidification of the culture medium. Thus, T cell proliferation can beevidenced by, for example, examining the size or measuring the volume ofthe T cells, such as with a Coulter Counter. A resting T cell has a meandiameter of about 6.8 microns. Following the initial activation andstimulation the T cell mean diameter will increase to over 12 microns byday 4 and begin to decrease by about day 6. Moreover, T cellproliferation can be assessed by standard techniques known in the art,such as tritiated thymidine uptake.

The method of the invention involves contacting proliferating T cellswith at least one stimulatory agent prior to introducing the nucleicacid molecule into the proliferating T cell. The term “stimulatoryagent” is intended to include agents which provide a signal to the Tcell, such that the level of expression of the gene comprised in thenucleic acid molecule transfected into the T cell is higher when the Tcell is contacted with the stimulatory agent prior to introducing thenucleic acid molecule into the T cell, than in T cells not contactedwith the stimulatory agent prior to introducing the nucleic acidmolecule.

In a specific embodiment of the invention, the stimulatory agent is anagent which provides a primary activation signal to a T cell. Thelanguage “primary activation signal” is intended to include signals,typically triggered through the TCR/CD3 complex, that induce activationof T cells. Activation of a T cell is intended to include modificationsof a T cell, such that the T cell is induced to proliferate anddifferentiate upon receiving a second signal, such as a costimulatorysignal. In a specific embodiment, the primary activation signal isprovided by an agent which contacts the T cell receptor or the CD3complex associated with the T cell receptor. In a preferred embodiment,the agent is an antibody reactive against CD3, such as the monoclonalantibody OKT3 (available from the American Type Culture Collection,Rockville, Md.; No. CRL 8001). In another embodiment of the invention,the stimulating agent is an agent that stimulates the CD2 complex on Tcells, such as a combination of antibodies, e.g. T11.3+T11.1 orT11.3+T11.2 (see e.g., Meuer et al., 1984).

In another embodiment of the method, the stimulatory agent is alymphokine, such as IL-2. The lymphokine is particularly used incombination with another agent, such as an agent which provides aprimary activation signal to the T cell, for stimulating T cells. Thus,in a preferred embodiment of the invention, T cells are contacted with acombination of an agent which provides a primary activation signal tothe T cells (e.g., an anti-CD3 antibody) and an effective amount ofIL-2, prior to transfecting the T cells with a nucleic acid molecule,such that the nucleic acid molecule is expressed in the T cells.

In a preferred embodiment of the invention, the T cells are activatedwith a combination of agents that stimulate both a primary activationsignal and a costimulatory signal in the T cell. The term “costimulatoryagent” is intended to include agents which provide a costimulatorysignal in T cells, such that a T cell that has received a primaryactivation signal (e.g. an activated T cell) is stimulated toproliferate or to secrete cytokines, such as IL-2, IL-4, orinterferon-γ. In a specific embodiment, the costimulatory agentinteracts with CD28 or CTLA4 molecules on the surface of the T cells. Inan even more specific embodiment, the costimulatory signal is a ligandof CD28 or CTLA4, such as a B-lymphocyte antigen B7-1 or B7-2. Thelanguage “stimulatory form of a natural ligand of CD28” is intended toinclude B7-1 and B7-2 molecules, fragments thereof, or modificationsthereof, which are capable of providing costimulatory signals to the Tcells. Stimulatory forms of natural ligands of CD28 can be identifiedby, for example, contacting activated peripheral blood lymphocytes witha form of a natural ligand of CD28 and performing a standard T cellproliferation assay. Thus, a stimulatory form of a natural ligand ofCD28 is capable of stimulating proliferation of the T cells. Stimulatoryforms of natural ligands of CD28/CTLA4 are described, for example, inPCT Publication No. WO 95/03408.

Other agents that can be used to activate T cells prior to introducing anucleic acid molecule into the T cell include agents that stimulate oneor more intracellular signal transduction pathways involved in T cellactivation and/or costimulation. In a specific embodiment of theinvention, the stimulatory agent is a calcium ionophore, such asionomycin or A23187. Alternatively, the stimulatory agent can be anagent which stimulates protein kinase C, such as a phorbol ester. Apreferred phorbol ester is phorbol-12,13-dibutyrate. In an even morepreferred embodiment of the invention, T cells are contacted with acombination of a calcium ionophore and a phorbol ester prior totransfection with a nucleic acid molecule. The stimulatory agent canalso be an agent which activates protein tyrosine kinases. A preferredagent that stimulates protein tyrosine kinases is pervanadate (O'Shea etal., 1992).

In yet another embodiment of the invention, the stimulatory agent is apolyclonal activator. Polyclonal activators include agents that bind toglycoproteins expressed on the plasma membrane of T cells and includelectins, such as phytohemaglutinin (PHA), concanavalin (Con A) andpokeweed mitogen (PWM).

By providing a clone a specific activation signal, it is possible toselectively transfect only a certain clone of T cells in a population ofT cells. Specific activation of a T cell clone can be accomplished, forexample, using a specific antigen presented by an antigen-presentingcell.

Other stimulating agents that can be used include super-antigens. Theterm “super-antigen” as defined herein is intended to include bacterialenterotoxins, or other bacterial proteins capable of stimulatingproliferation of T cells. Super-antigens include staphylococcalenterotoxins (SE), such as SEA, SEB, SEC, SED, and SEE. Super-antigenscan also be of viral origin, such as retroviral super-antigens.

Additional agents that are capable of stimulating T cells, either aloneor in combination with other agents, that may be identified using T cellstimulation assays as known in the art or described herein are alsowithin the scope of the invention. For stimulating T cells prior tointroduction of a nucleic acid molecule into the T cells, anycombination of the above described agents can be used.

The stimulating agents can be used in solution, or attached to a solidsurface. The solid surface can be, for example, the surface of a tissueculture dish or a bead. Depending on the nature of the stimulatoryagent, linkage to the solid surface can be performed by methods wellknown in the art. For example, proteins can be chemically crosslinked tothe cell surface using commercially available crosslinking reagents(Pierce, Rockford Ill.) or immobilized on plastic by overnightincubation at 4° C. If several agents are used for stimulation of the Tcells, some agents may be in solution and some agents may be attached toa solid support. In a preferred embodiment, the T cells are stimulatedwith a combination of solid phase coupled anti-CD3 antibody and solubleanti-CD28 antibody.

The specific doses of stimulatory agent(s) to be added to the T cellswill vary with the type of stimulating agent. Typically, the stimulatingagents are used at the same doses at which they are used for stimulatingT cells to proliferate and secrete cytokines, as described in the art.

In a preferred embodiment of the invention, the method of the inventionfurther comprises stimulating the T cells to expand in vitro aftertransfection of the T cells. T cells can be stimulated to expand invitro as described in the Examples section in the presence of IL-2. In aspecific embodiment, T cells may also be incubated with an agent whichprovides a primary activating signal, such as anti-CD3 and an agentwhich provides a costimulatory signal, such as an anti-CD28 antibody.

In an even more preferred embodiment, the T cells are primary T cells.Thus, T cells can be obtained from a subject, transfected according tothe method of the invention, and expanded in vitro. In anotherembodiment of the invention, the transfected and expanded T cells arere-administered to the subject. It may be preferable to further purifythe T cells prior to administering into the subject, such as by gradientcentrifugation.

E. V(D)J Recombination

The inventors discovered that V(D)J recombination in the T cellreceptors did not inhibit T cell reprogramming. The specificrearrangements of iPS cells derived from T cells may serve as a unique“bar code” to track iPS cells and identify different populations of iPScells in certain aspects. In a further aspect, there may also beprovided iPS cells with an incomplete set of V, D, J gene segments, ascompared with embryonic stem cells which have the original set of V, D,J gene segments. The arrangement of the V, D, J gene segments of theseiPS cells may be the same within a clonal population, but may bedifferent among different clonal populations. In particular aspects,gamma/delta TCR⁺ T-cells may be also reprogrammed with the presentmethods. An iPS clone originating from one of this population of T-cellscould be advantageous because they may have a genome that more closelyresembles the germ line configuration and thus may be able tore-differentiate into a more robust repertoire of T-cells or otherdifferentiated cells, for example.

V(D)J recombination is a mechanism of genetic recombination that occursin vertebrates, which randomly selects and assembles segments of genesencoding specific proteins with important roles in the immune system.This site-specific recombination reaction generates a diverse repertoireof T cell receptor (TCR) and immunoglobulin (Ig) molecules that arenecessary for the recognition of diverse antigens from bacterial, viral,and parasitic invaders, and from dysfunctional cells such as tumorcells.

Most T cell receptors are composed of an alpha chain and a beta chain.The T cell receptor genes are similar to immunoglobulin genes in thatthey too contain multiple V, D and J genes in their beta chains (and Vand J genes in their alpha chains) that are rearranged during thedevelopment of the lymphocyte to provide that cell with a unique antigenreceptor.

During T cell development, the T cell receptor (TCR) chains undergoessentially the same sequence of ordered recombination events as thatdescribed for immunoglobulins. D-to-J recombination occurs first in theβ chain of the TCR. This process can involve either the joining of theD_(β)1 gene segment to one of six J_(β)1 segments or the joining of theD_(β)2 gene segment to one of six J_(β)2 segments. DJ recombination isfollowed (as above) with V_(β)-to-D_(β)J_(β) rearrangements. All genesbetween the V_(β)-D_(β)-J_(β) genes in the newly formed complex aredeleted and the primary transcript is synthesized that incorporates theconstant domain gene (V_(β)-D_(β)-J_(β)−C_(β)). mRNA transcriptionsplices out any intervening sequence and allows translation of the fulllength protein for the TCR C_(β) chain.

The rearrangement of the alpha (α) chain of the TCR follows β chainrearrangement, and resembles V-to-J rearrangement described for Ig lightchains (see above). The assembly of the β- and α-chains results information of the αβ-TCR that is expressed on a majority of T cells.

VI. Reprogramming Factors Expression and Transduction

In certain aspects of the present invention, reprogramming factors areexpressed from expression cassettes comprised in one or more vectors,such as an integrating vector or an episomal vector. In a furtheraspect, reprogramming proteins could be introduced directly into somaticcells by protein transduction (see U.S. Application No. 61/172,079,incorporated herein by reference).

A. Integrating Vectors

IPS cells may be derived by transfection of certain nucleic acids orgenes encoding reprogramming proteins into non-pluripotent cells, suchas T cells or hematopoietic precursor cells, in the present invention.Transfection is typically achieved through integrating viral vectors inthe current practice, such as retroviruses. Transfected genes mayinclude the master transcriptional regulators Oct4 (Pouf51) and Sox2,although it is suggested that other genes enhance the efficiency ofinduction. After a critical period, small numbers of transfected cellsmay begin to become morphologically and biochemically similar topluripotent stem cells, and could be isolated through morphologicalselection, doubling time, or through a reporter gene and antibioticinfection.

In November 2007, a milestone was achieved by creating iPS from adulthuman fibroblasts from two independent research teams' studies (Yu etal., 2007; Yamanaka et al., 2007). With the same principle used earlierin mouse models, Yamanaka had successfully transformed human fibroblastsinto pluripotent stem cells using the same four pivotal genes: Oct4,Sox2, Klf4, and c-Myc with a retroviral system but c-Myc is oncogenic.Thomson and colleagues used Oct4, Sox2, NANOG, and a different geneLIN28 using a lentiviral system avoiding the use of c-Myc. Morerecently, fertile mice have been generated from iPS cells, thusdemonstrating the potential of these cells to form essentially any orall differentiated cell types (Boland et al., 2009).

As described above, induction of pluripotent stem cells from humandermal fibroblasts has been achieved using retroviruses or lentiviralvectors for ectopic expression of reprogramming genes. Recombinantretroviruses such as the Moloney murine leukemia virus have the abilityto integrate into the host genome in a stable fashion. They contain areverse transcriptase which allows integration into the host genome.Lentiviruses are a subclass of Retroviruses. They are widely adapted asvectors thanks to their ability to integrate into the genome ofnon-dividing as well as dividing cells. The viral genome in the form ofRNA is reverse-transcribed when the virus enters the cell to produceDNA, which is then inserted into the genome at a random position by theviral integrase enzyme. Therefore, successful reprogramming of T cellsmay use integration-based viral approaches as shown in the Examplessection.

B. Episomal Vectors

These reprogramming methods may also make use of extra-chromosomallyreplicating vectors (i.e., episomal vectors), which are vectors capableof replicating episomally to make iPS cells essentially free ofexogenous vector or viral elements (see U.S. Application No. 61/058,858,incorporated herein by reference; Yu et al., 2009). A number of DNAviruses, such as adenoviruses, Simian vaculating virus 40 (SV40) orbovine papilloma virus (BPV), or budding yeast ARS (AutonomouslyReplicating Sequences)-containing plasmids replicate extra-chromosomallyor episomally in mammalian cells. These episomal plasmids areintrinsically free from all these disadvantages (Bode et al., 2001)associated with integrating vectors. For example, a lymphotrophic herpesvirus-based including or Epstein Ban Virus (EBV) as defined above mayreplicate extra-chromosomally and help deliver reprogramming genes tosomatic cells.

For example, the plasmid-based approach used in the invention mayextract robust elements necessary for the successful replication andmaintenance of an EBV element-based system without compromising thesystem's tractability in a clinical setting as described in detailbelow. The essential EBV elements are OriP and EBNA-1 or their variantsor functional equivalents. An additional advantage of this system isthat these exogenous elements will be lost with time after beingintroduced into cells, leading to self-sustained iPS cells essentiallyfree of exogenous elements.

The use of plasmid- or liposome-based extra-chromosomal vectors, e.g.,oriP-based vectors, and/or vectors encoding a derivative of EBNA-1permit large fragments of DNA to be introduced to a cell and maintainedextra-chromosomally, replicated once per cell cycle, partitioned todaughter cells efficiently, and elicit substantially no immune response.In particular, EBNA-1, the only viral protein required for thereplication of the oriP-based expression vector, does not elicit acellular immune response because it has developed an efficient mechanismto bypass the processing required for presentation of its antigens onMHC class I molecules (Levitskaya et al., 1997). Further, EBNA-1 can actin trans to enhance expression of the cloned gene, inducing expressionof a cloned gene up to 100-fold in some cell lines (Langle-Rouault etal., 1998; Evans et al., 1997). Finally, the manufacture of suchoriP-based expression vectors is inexpensive.

Other extra-chromosomal vectors include other lymphotrophic herpesvirus-based vectors. Lymphotrophic herpes virus is a herpes virus thatreplicates in a lymphoblast (e.g., a human B lymphoblast) and becomes aplasmid for a part of its natural life-cycle. Herpes simplex virus (HSV)is not a “lymphotrophic” herpes virus. Exemplary lymphotrophic herpesviruses include, but are not limited to EBV, Kaposi's sarcoma herpesvirus (KSHV); Herpes virus saimiri (HS) and Marek's disease virus (MDV).Also other sources of episome-base vectors are contemplated, such asyeast ARS, adenovirus, SV40, or BPV.

To circumvent potential problems from viral gene delivery, two groupsthis year reported on a collaboration that has succeeded intransposon-based approaches for producing pluripotency in human cellswithout using viral vectors (Woltjen et al., 2009; Kaji et al., 2009).Stable iPS cells were produced in both human and mouse fibroblasts usingvirus-derived 2A peptide sequences to create a multicistronic vectorincorporating the reprogramming factors, delivered to the cell by thepiggyBac transposon vector. The 2A-linked reprogramming factors, notrequired in the established iPS cell lines, were then removed. Thesestrategies could be similarly applied to reprogram T cells orhematopoietic precursor cells in certain aspects of the presentinvention.

C. Protein Transduction

One possible way to avoid introducing exogenous genetic modifications totarget cells would be to deliver the reprogramming proteins directlyinto cells, rather than relying on the transcription from deliveredgenes. Previous studies have demonstrated that various proteins can bedelivered into cells in vitro and in vivo by conjugating them with ashort peptide that mediates protein transduction, such as HIV tat andpoly-arginine. A recent study demonstrated that murine fibroblasts canbe fully reprogrammed into pluripotent stem cells by direct delivery ofrecombinant reprogramming proteins (Zhou et al., 2009). More details ofthe methods for reprogramming cells with protein transduction have beendisclosed in U.S. Application No. 61/172,079 incorporated herein byreference.

In certain aspects of the present invention, protein transductiondomains could been used to introduce reprogramming proteins directlyinto T cells. Protein transduction may be used to enhance the deliveryof reprogramming proteins into cells. For example, a region of the TATprotein which is derived from the HIV Tat protein can be fused to atarget protein allowing the entry of the target protein into the cell.The advantages of using fusions of these transduction domains is thatprotein entry is rapid, concentration-dependent and appears to work withdifferent cell types.

In a further aspect of the present invention, a nuclear localizationsequence may also be used to facilitate nuclear entry of reprogrammingproteins. Nuclear localization signals (NLS) have been described forvarious proteins. The mechanism of protein transport to the nucleus isthrough the binding of a target protein containing a nuclearlocalization signal to alpha subunit of karyopherin. This is followed bytransport of the target protein:karyopherin complex through the nuclearpore and into the nucleus. However, reprogramming proteins are oftentranscription factors which may have endogenous nuclear localizationsequences. Therefore, nuclear localization sequences may not benecessary.

The direct introduction of reprogramming proteins into somatic cells maybe used in the present invention, with reprogramming proteinsoperatively linked to a protein transduction domain (PTD), either bycreating a fusion protein comprising such a domain or by chemicallycross-linking the reprogramming protein and PTD via functional groups oneach molecule.

Standard recombinant nucleic acid methods can be used to express one ormore transducible reprogramming proteins used herein. In one embodiment,a nucleic acid sequence encoding the transducible protein is cloned intoa nucleic acid expression vector, e.g., with appropriate signal andprocessing sequences and regulatory sequences for transcription andtranslation. In another embodiment, the protein can be synthesized usingautomated organic synthetic methods.

In addition, there have been several methods that may also help thetransport of proteins into cells, one ore more of which can be usedalone or in combination with the methods using the protein transductiondomains, including, but not limited to, microinjection, electroporation,and the use of liposomes. Most of these methods may need a purifiedpreparation of protein. Purification of recombinant proteins is oftenfacilitated by the incorporation of an affinity tag into the expressionconstruct, making the purification step fast and efficient.

VII. Vector Construction and Delivery

In certain embodiments, reprogramming vectors could be constructed tocomprise additional elements in addition to nucleic acid sequencesencoding reprogramming factors as described above in cells. Details ofcomponents of these vectors and delivery methods are disclosed below.

A. Vector

One of skill in the art would be well equipped to construct a vectorthrough standard recombinant techniques (see, for example, Maniatis etal., 1988 and Ausubel et al., 1994, both incorporated herein byreference).

Vectors can also comprise other components or functionalities thatfurther modulate gene delivery and/or gene expression, or that otherwiseprovide beneficial properties to the targeted cells. Such othercomponents include, for example, components that influence binding ortargeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide.

Such components also might include markers, such as detectable and/orselection markers that can be used to detect or select for cells thathave taken up and are expressing the nucleic acid delivered by thevector. Such components can be provided as a natural feature of thevector (such as the use of certain viral vectors which have componentsor functionalities mediating binding and uptake), or vectors can bemodified to provide such functionalities. A large variety of suchvectors are known in the art and are generally available. When a vectoris maintained in a host cell, the vector can either be stably replicatedby the cells during mitosis as an autonomous structure, incorporatedwithin the genome of the host cell, or maintained in the host cell'snucleus or cytoplasm.

B. Regulatory Elements

Eukaryotic expression cassettes included in the vectors particularlycontain (in a 5′-to-3′ direction) a eukaryotic transcriptional promoteroperably linked to a protein-coding sequence, splice signals includingintervening sequences, and a transcriptional termination/polyadenylationsequence.

i. Promoter/Enhancers

A “promoter” is a control sequence that is a region of a nucleic acidsequence at which initiation and rate of transcription are controlled.It may contain genetic elements at which regulatory proteins andmolecules may bind, such as RNA polymerase and other transcriptionfactors, to initiate the specific transcription a nucleic acid sequence.The phrases “operatively positioned,” “operatively linked,” “undercontrol,” and “under transcriptional control” mean that a promoter is ina correct functional location and/or orientation in relation to anucleic acid sequence to control transcriptional initiation and/orexpression of that sequence.

Promoters suitable for use in EBNA 1-encoding vector of the inventionare those that direct the expression of the expression cassettesencoding the EBNA 1 protein to result in sufficient steady-state levelsof EBNA 1 protein to stably maintain EBV oriP-containing vectors.Promoters are also used for efficient expression of expression cassettesencoding reprogramming factors.

A promoter generally comprises a sequence that functions to position thestart site for RNA synthesis. The best known example of this is the TATAbox, but in some promoters lacking a TATA box, such as, for example, thepromoter for the mammalian terminal deoxynucleotidyl transferase geneand the promoter for the SV40 late genes, a discrete element overlyingthe start site itself helps to fix the place of initiation. Additionalpromoter elements regulate the frequency of transcriptional initiation.Typically, these are located in the region 30-110 bp upstream of thestart site, although a number of promoters have been shown to containfunctional elements downstream of the start site as well. To bring acoding sequence “under the control of” a promoter, one positions the 5′end of the transcription initiation site of the transcriptional readingframe “downstream” of (i.e., 3′ of) the chosen promoter. The “upstream”promoter stimulates transcription of the DNA and promotes expression ofthe encoded RNA.

The spacing between promoter elements frequently is flexible, so thatpromoter function is preserved when elements are inverted or movedrelative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription. A promoter may or may not be used in conjunction with an“enhancer,” which refers to a cis-acting regulatory sequence involved inthe transcriptional activation of a nucleic acid sequence.

A promoter may be one naturally associated with a nucleic acid sequence,as may be obtained by isolating the 5′ non-coding sequences locatedupstream of the coding segment and/or exon. Such a promoter can bereferred to as “endogenous.” Similarly, an enhancer may be one naturallyassociated with a nucleic acid sequence, located either downstream orupstream of that sequence. Alternatively, certain advantages will begained by positioning the coding nucleic acid segment under the controlof a recombinant or heterologous promoter, which refers to a promoterthat is not normally associated with a nucleic acid sequence in itsnatural environment. A recombinant or heterologous enhancer refers alsoto an enhancer not normally associated with a nucleic acid sequence inits natural environment. Such promoters or enhancers may includepromoters or enhancers of other genes, and promoters or enhancersisolated from any other virus, or prokaryotic or eukaryotic cell, andpromoters or enhancers not “naturally occurring,” i.e., containingdifferent elements of different transcriptional regulatory regions,and/or mutations that alter expression. For example, promoters that aremost commonly used in recombinant DNA construction include theβ-lactamase (penicillinase), lactose and tryptophan (trp) promotersystems. In addition to producing nucleic acid sequences of promotersand enhancers synthetically, sequences may be produced using recombinantcloning and/or nucleic acid amplification technology, including PCR™, inconnection with the compositions disclosed herein (see U.S. Pat. Nos.4,683,202 and 5,928,906, each incorporated herein by reference).Furthermore, it is contemplated the control sequences that directtranscription and/or expression of sequences within non-nuclearorganelles such as mitochondria, chloroplasts, and the like, can beemployed as well.

Naturally, it will be important to employ a promoter and/or enhancerthat effectively directs the expression of the DNA segment in theorganelle, cell type, tissue, organ, or organism chosen for expression.Those of skill in the art of molecular biology generally know the use ofpromoters, enhancers, and cell type combinations for protein expression,(see, for example Sambrook et al. 1989, incorporated herein byreference). The promoters employed may be constitutive, tissue-specific,inducible, and/or useful under the appropriate conditions to direct highlevel expression of the introduced DNA segment, such as is advantageousin the large-scale production of recombinant proteins and/or peptides.The promoter may be heterologous or endogenous.

Additionally any promoter/enhancer combination (as per, for example, theEukaryotic Promoter Data Base EPDB, through world wide web atepd.isb-sib.ch/) could also be used to drive expression. Use of a T3, T7or SP6 cytoplasmic expression system is another possible embodiment.Eukaryotic cells can support cytoplasmic transcription from certainbacterial promoters if the appropriate bacterial polymerase is provided,either as part of the delivery complex or as an additional geneticexpression construct.

Non-limiting examples of promoters include early or late viralpromoters, such as, SV40 early or late promoters, cytomegalovirus (CMV)immediate early promoters, Rous Sarcoma Virus (RSV) early promoters;eukaryotic cell promoters, such as, e.g., beta actin promoter (Ng, 1989;Quitsche et al., 1989), GADPH promoter (Alexander et al., 1988, Ercolaniet al., 1988), metallothionein promoter (Karin et al., 1989; Richards etal., 1984); and concatenated response element promoters, such as cyclicAMP response element promoters (cre), serum response element promoter(sre), phorbol ester promoter (TPA) and response element promoters (tre)near a minimal TATA box. It is also possible to use human growth hormonepromoter sequences (e.g., the human growth hormone minimal promoterdescribed at Genbank, accession no. X05244, nucleotide 283-341) or amouse mammary tumor promoter (available from the ATCC, Cat. No. ATCC45007). A specific example could be a phosphoglycerate kinase (PGK)promoter.

ii. Protease Cleavage Sites/self-cleaving Peptides and Internal RibosomeBinding Sites

In certain aspects, according to the present invention, the genesencoding markers or reprogramming proteins may be connected to oneanother by a sequence (there may be more than one) coding for a proteasecleavage site (i.e. a sequence comprising the recognition site of aprotease) or at least one self-cleaving peptide.

According to a certain embodiment of the present invention theprotease(s) capable of cleaving the cleavage sites encoded by thesequence(s) connecting the genes constituting the polycistronic messageis/are encoded by the polynucleotide of the present invention. Moreparticularly, the gene(s) encoding the protease(s) is/are part of atleast one of the polycistronic message.

Suitable protease cleavages sites and self-cleaving peptides are knownto the skilled person (see, e.g., in Ryan et al., 1997; Scymczak et al.,2004). Preferred examples of protease cleavage sites are the cleavagesites of potyvirus NIa proteases (e.g. tobacco etch virus protease),potyvirus HC proteases, potyvirus P1 (P35) proteases, byovirus N1aproteases, byovirus RNA-2-encoded proteases, aphthovirus L proteases,enterovirus 2A proteases, rhinovirus 2A proteases, picorna 3C proteases,comovirus 24K proteases, nepovirus 24K proteases, RTSV (rice tungrospherical virus) 3Ciike protease, PY\IF (parsnip yellow fleck virus)3C-like protease, thrombin, factor Xa and enterokinase. Due to its highcleavage stringency, TEV (tobacco etch virus) protease cleavage sitesmay be used.

Exemplary self-cleaving peptides (also called “cis-acting hydrolyticelements”, CHYSEL; see deFelipe (2002) are derived from potyvirus andcardiovirus 2A peptides. Particular self-cleaving peptides may beselected from 2A peptides derived from FMDV (foot-and-mouth diseasevirus), equine rhinitis A virus, Thoseà asigna virus and porcineteschovirus.

A specific initiation signal also may be used for efficient translationof coding sequences in a polycistronic message. These signals includethe ATG initiation codon or adjacent sequences. Exogenous translationalcontrol signals, including the ATG initiation codon, may need to beprovided. One of ordinary skill in the art would readily be capable ofdetermining this and providing the necessary signals. It is well knownthat the initiation codon must be “in-frame” with the reading frame ofthe desired coding sequence to ensure translation of the entire insert.The exogenous translational control signals and initiation codons can beeither natural or synthetic. The efficiency of expression may beenhanced by the inclusion of appropriate transcription enhancerelements.

In certain embodiments of the invention, the use of internal ribosomeentry sites (IRES) elements are used to create multigene, orpolycistronic, messages. IRES elements are able to bypass the ribosomescanning model of 5′ methylated Cap dependent translation and begintranslation at internal sites (Pelletier and Sonenberg, 1988). IRESelements from two members of the picornavirus family (polio andencephalomyocarditis) have been described (Pelletier and Sonenberg,1988), as well an IRES from a mammalian message (Macejak and Sarnow,1991). IRES elements can be linked to heterologous open reading frames.Multiple open reading frames can be transcribed together, each separatedby an IRES, creating polycistronic messages. By virtue of the IRESelement, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message (see U.S. Pat.Nos. 5,925,565 and 5,935,819, each herein incorporated by reference).

iii. Multiple Cloning Sites

Vectors can include a multiple cloning site (MCS), which is a nucleicacid region that contains multiple restriction enzyme sites, any ofwhich can be used in conjunction with standard recombinant technology todigest the vector (see, for example, Carbonelli et al., 1999, Levensonet al., 1998, and Cocea, 1997, incorporated herein by reference.)“Restriction enzyme digestion” refers to catalytic cleavage of a nucleicacid molecule with an enzyme that functions only at specific locationsin a nucleic acid molecule. Many of these restriction enzymes arecommercially available. Use of such enzymes is widely understood bythose of skill in the art. Frequently, a vector is linearized orfragmented using a restriction enzyme that cuts within the MCS to enableexogenous sequences to be ligated to the vector. “Ligation” refers tothe process of forming phosphodiester bonds between two nucleic acidfragments, which may or may not be contiguous with each other.Techniques involving restriction enzymes and ligation reactions are wellknown to those of skill in the art of recombinant technology.

iv. Splicing Sites

Most transcribed eukaryotic RNA molecules will undergo RNA splicing toremove introns from the primary transcripts. Vectors containing genomiceukaryotic sequences may require donor and/or acceptor splicing sites toensure proper processing of the transcript for protein expression (see,for example, Chandler et al., 1997, herein incorporated by reference.)

v. Termination Signals

The vectors or constructs of the present invention will generallycomprise at least one termination signal. A “termination signal” or“terminator” is comprised of the DNA sequences involved in specifictermination of an RNA transcript by an RNA polymerase. Thus, in certainembodiments a termination signal that ends the production of an RNAtranscript is contemplated. A terminator may be necessary in vivo toachieve desirable message levels.

In eukaryotic systems, the terminator region may also comprise specificDNA sequences that permit site-specific cleavage of the new transcriptso as to expose a polyadenylation site. This signals a specializedendogenous polymerase to add a stretch of about 200 A residues (polyA)to the 3′ end of the transcript. RNA molecules modified with this polyAtail appear to more stable and are translated more efficiently. Thus, inother embodiments involving eukaryotes, it is preferred that thatterminator comprises a signal for the cleavage of the RNA, and it ismore preferred that the terminator signal promotes polyadenylation ofthe message. The terminator and/or polyadenylation site elements canserve to enhance message levels and to minimize read through from thecassette into other sequences.

Terminators contemplated for use in the invention include any knownterminator of transcription described herein or known to one of ordinaryskill in the art, including but not limited to, for example, thetermination sequences of genes, such as for example the bovine growthhormone terminator or viral termination sequences, such as for examplethe SV40 terminator. In certain embodiments, the termination signal maybe a lack of transcribable or translatable sequence, such as due to asequence truncation.

vi. Polyadenylation Signals

In expression, particularly eukaryotic expression, one will typicallyinclude a polyadenylation signal to effect proper polyadenylation of thetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed. Preferred embodiments include the SV40polyadenylation signal or the bovine growth hormone polyadenylationsignal, convenient and known to function well in various target cells.Polyadenylation may increase the stability of the transcript or mayfacilitate cytoplasmic transport.

vii. Origins of Replication

In order to propagate a vector in a host cell, it may contain one ormore origins of replication sites (often termed “ori”), for example, anucleic acid sequence corresponding to oriP of EBV as described above ora genetically engineered oriP with a similar or elevated function indifferentiation programming, which is a specific nucleic acid sequenceat which replication is initiated. Alternatively a replication origin ofother extra-chromosomally replicating virus as described above or anautonomously replicating sequence (ARS) can be employed.

viii. Selection and Screenable Markers

In certain embodiments of the invention, cells containing a nucleic acidconstruct of the present invention may be identified in vitro or in vivoby including a marker in the expression vector. Such markers wouldconfer an identifiable change to the cell permitting easy identificationof cells containing the expression vector. Generally, a selection markeris one that confers a property that allows for selection. A positiveselection marker is one in which the presence of the marker allows forits selection, while a negative selection marker is one in which itspresence prevents its selection. An example of a positive selectionmarker is a drug resistance marker.

Usually the inclusion of a drug selection marker aids in the cloning andidentification of transformants, for example, genes that conferresistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin andhistidinol are useful selection markers. In addition to markersconferring a phenotype that allows for the discrimination oftransformants based on the implementation of conditions, other types ofmarkers including screenable markers such as GFP, whose basis iscolorimetric analysis, are also contemplated. Alternatively, screenableenzymes as negative selection markers such as herpes simplex virusthymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may beutilized. One of skill in the art would also know how to employimmunologic markers, possibly in conjunction with FACS analysis. Themarker used is not believed to be important, so long as it is capable ofbeing expressed simultaneously with the nucleic acid encoding a geneproduct. Further examples of selection and screenable markers are wellknown to one of skill in the art. One feature of the present inventionincludes using selection and screenable markers to select vector-freecells after the differentiation programming factors have effected adesired altered differentiation status in those cells.

C. Vector Delivery

Introduction of a reprogramming vector into somatic cells with thecurrent invention may use any suitable methods for nucleic acid deliveryfor transformation of a cell, as described herein or as would be knownto one of ordinary skill in the art. Such methods include, but are notlimited to, direct delivery of DNA such as by ex vivo transfection(Wilson et al., 1989, Nabel et al, 1989), by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, 1985; U.S.Pat. No. 5,789,215, incorporated herein by reference); byelectroporation (U.S. Pat. No. 5,384,253, incorporated herein byreference; Tur-Kaspa et al., 1986; Potter et al., 1984); by calciumphosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama,1987; Rippe et al., 1990); by using DEAE-dextran followed bypolyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimeret al., 1987); by liposome mediated transfection (Nicolau and Sene,1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980;Kaneda et al., 1989; Kato et al., 1991) and receptor-mediatedtransfection (Wu and Wu, 1987; Wu and Wu, 1988); by microprojectilebombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat.Nos. 5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880,and each incorporated herein by reference); by agitation with siliconcarbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and5,464,765, each incorporated herein by reference); byAgrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and5,563,055, each incorporated herein by reference); by PEG-mediatedtransformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos.4,684,611 and 4,952,500, each incorporated herein by reference); bydesiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985), andany combination of such methods. Through the application of techniquessuch as these, organelle(s), cell(s), tissue(s) or organism(s) may bestably or transiently transformed.

i. Liposome-Mediated Transfection

In a certain embodiment of the invention, a nucleic acid may beentrapped in a lipid complex such as, for example, a liposome. Liposomesare vesicular structures characterized by a phospholipid bilayermembrane and an inner aqueous medium. Multilamellar liposomes havemultiple lipid layers separated by aqueous medium. They formspontaneously when phospholipids are suspended in an excess of aqueoussolution. The lipid components undergo self-rearrangement before theformation of closed structures and entrap water and dissolved solutesbetween the lipid bilayers (Ghosh and Bachhawat, 1991). Alsocontemplated is an nucleic acid complexed with Lipofectamine (Gibco BRL)or Superfect (Qiagen). The amount of liposomes used may vary upon thenature of the liposome as well as the, cell used, for example, about 5to about 20 μg vector DNA per 1 to 10 million of cells may becontemplated.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful (Nicolau and Sene, 1982; Fraley et al.,1979; Nicolau et al., 1987). The feasibility of liposome-mediateddelivery and expression of foreign DNA in cultured chick embryo, HeLaand hepatoma cells has also been demonstrated (Wong et al., 1980).

In certain embodiments of the invention, a liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, aliposome may be complexed or employed in conjunction with nuclearnon-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, a liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In other embodiments, a deliveryvehicle may comprise a ligand and a liposome.

ii. Electroporation

In certain embodiments of the present invention, a nucleic acid isintroduced into a cell via electroporation. Electroporation involves theexposure of a suspension of cells and DNA to a high-voltage electricdischarge. Recipient cells can be made more susceptible totransformation by mechanical wounding. Also the amount of vectors usedmay vary upon the nature of the cells used, for example, about 5 toabout 20 μg vector DNA per 1 to 10 million of cells may be contemplated.

Transfection of eukaryotic cells using electroporation has been quitesuccessful. Mouse pre-B lymphocytes have been transfected with humankappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocyteshave been transfected with the chloramphenicol acetyltransferase gene(Tur-Kaspa et al., 1986) in this manner.

iii. Calcium Phosphate

In other embodiments of the present invention, a nucleic acid isintroduced to the cells using calcium phosphate precipitation. Human KBcells have been transfected with adenovirus 5 DNA (Graham and Van DerEb, 1973) using this technique. Also in this manner, mouse L(A9), mouseC127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with aneomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes weretransfected with a variety of marker genes (Rippe et al., 1990).

iv. DEAE-Dextran

In another embodiment, a nucleic acid is delivered into a cell usingDEAE-dextran followed by polyethylene glycol. In this manner, reporterplasmids were introduced into mouse myeloma and erythroleukemia cells(Gopal, 1985).

v. Sonication Loading

Additional embodiments of the present invention include the introductionof a nucleic acid by direct sonic loading. LTK⁻ fibroblasts have beentransfected with the thymidine kinase gene by sonication loading(Fechheimer et al., 1987).

vi. Receptor Mediated Transfection

Still further, a nucleic acid may be delivered to a target cell viareceptor-mediated delivery vehicles. These take advantage of theselective uptake of macromolecules by receptor-mediated endocytosis thatwill be occurring in a target cell. In view of the cell type-specificdistribution of various receptors, this delivery method adds anotherdegree of specificity to the present invention.

Certain receptor-mediated gene targeting vehicles comprise a cellreceptor-specific ligand and a nucleic acid-binding agent. Otherscomprise a cell receptor-specific ligand to which the nucleic acid to bedelivered has been operatively attached. Several ligands have been usedfor receptor-mediated gene transfer (Wu and Wu, 1987; Wagner et al.,1990; Perales et al., 1994; Myers, EPO 0273085), which establishes theoperability of the technique. Specific delivery in the context ofanother mammalian cell type has been described (Wu and Wu, 1993;incorporated herein by reference). In certain aspects of the presentinvention, a ligand will be chosen to correspond to a receptorspecifically expressed on the target cell population.

In other embodiments, a nucleic acid delivery vehicle component of acell-specific nucleic acid targeting vehicle may comprise a specificbinding ligand in combination with a liposome. The nucleic acid(s) to bedelivered are housed within the liposome and the specific binding ligandis functionally incorporated into the liposome membrane. The liposomewill thus specifically bind to the receptor(s) of a target cell anddeliver the contents to a cell. Such systems have been shown to befunctional using systems in which, for example, epidermal growth factor(EGF) is used in the receptor-mediated delivery of a nucleic acid tocells that exhibit upregulation of the EGF receptor.

In still further embodiments, the nucleic acid delivery vehiclecomponent of a targeted delivery vehicle may be a liposome itself, whichwill particularly comprise one or more lipids or glycoproteins thatdirect cell-specific binding. For example, lactosyl-ceramide, agalactose-terminal asialganglioside, have been incorporated intoliposomes and observed an increase in the uptake of the insulin gene byhepatocytes (Nicolau et al., 1987). It is contemplated that thetissue-specific transforming constructs of the present invention can bespecifically delivered into a target cell in a similar manner.

vii Microprojectile Bombardment

Microprojectile bombardment techniques can be used to introduce anucleic acid into at least one, organelle, cell, tissue or organism(U.S. Pat. Nos. 5,550,318; 5,538,880; 5,610,042; and PCT Application WO94/09699; each of which is incorporated herein by reference). Thismethod depends on the ability to accelerate DNA-coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). There are a widevariety of microprojectile bombardment techniques known in the art, manyof which are applicable to the invention.

In this microprojectile bombardment, one or more particles may be coatedwith at least one nucleic acid and delivered into cells by a propellingforce. Several devices for accelerating small particles have beendeveloped. One such device relies on a high voltage discharge togenerate an electrical current, which in turn provides the motive force(Yang et al., 1990). The microprojectiles used have consisted ofbiologically inert substances such as tungsten or gold particles orbeads. Exemplary particles include those comprised of tungsten,platinum, and particularly, gold. It is contemplated that in someinstances DNA precipitation onto metal particles would not be necessaryfor DNA delivery to a recipient cell using microprojectile bombardment.However, it is contemplated that particles may contain DNA rather thanbe coated with DNA. DNA-coated particles may increase the level of DNAdelivery via particle bombardment but are not, in and of themselves,necessary.

For the bombardment, cells in suspension are concentrated on filters orsolid culture medium. Alternatively, immature embryos or other targetcells may be arranged on solid culture medium. The cells to be bombardedare positioned at an appropriate distance below the macroprojectilestopping plate.

VIII. Selection of iPS Cells

In certain aspects of the invention, after one or more reprogrammingfactors are introduced into somatic cells, cells will be cultured forexpansion (optionally selected for the presence of vector elements likepositive selection or screenable marker to concentrate transfectedcells). Reprogramming vectors may express reprogramming factors in thesecells and replicate and partition along with cell division.Alternatively, reprogramming proteins could enter these cells and theirprogeny by replenishing medium containing the reprogramming proteins.These reprogramming factors will reprogram somatic cell genome toestablish a self-sustaining pluripotent state, and in the meantime orafter removal of positive selection of the presence of vectors,exogenous genetic elements will be lost gradually, or there is no needto add reprogramming proteins.

These induced pluripotent stem cells could be selected from progenyderived from these T cells or hematopoietic precursor cells based onembryonic stem cell characteristics because they are expected to besubstantially identical to pluripotent embryonic stem cells. Anadditional negative selection step could be also employed to accelerateor help selection of iPS cells essentially free of exogenous geneticelements by testing the absence of reprogramming vector DNA or usingselection markers, such as reporters.

A. Selection for Embryonic Stem Cell Characteristics

The successfully generated iPSCs from previous studies were remarkablysimilar to naturally-isolated pluripotent stem cells (such as mouse andhuman embryonic stem cells, mESCs and hESCs, respectively) in thefollowing respects, thus confirming the identity, authenticity, andpluripotency of iPSCs to naturally-isolated pluripotent stem cells.Thus, induced pluripotent stem cells generated from the methodsdisclosed in this invention could be selected based on one or more offollowing embryonic stem cell characteristics.

i. Cellular Biological Properties

Morphology: iPSCs are morphologically similar to ESCs. Each cell mayhave round shape, dual nucleoli or large nucleolus and scant cytoplasm.Colonies of iPSCs could be also similar to that of ESCs. Human iPSCsform sharp-edged, flat, tightly-packed colonies similar to hESCs andmouse iPSCs form the colonies similar to mESCs, less flat and moreaggregated colonies than that of hESCs.

Growth Properties: Doubling time and mitotic activity are cornerstonesof ESCs, as stem cells must self-renew as part of their definition.iPSCs could be mitotically active, actively self-renewing,proliferating, and dividing at a rate equal to ESCs.

Stem Cell Markers: iPSCs may express cell surface antigenic markersexpressed on ESCs. Human iPSCs expressed the markers specific to hESC,including, but not limited to, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81,TRA-2-49/6E, and Nanog. Mouse iPSCs expressed SSEA-1 but not SSEA-3 norSSEA-4, similarly to mESCs.

Stem Cell Genes: iPSCs may express genes expressed in undifferentiatedESCs, including Oct4, Sox2, Nanog, GDF3, REX 1, FGF4, ESG1, DPPA2,DPPA4, and hTERT.

Telomerase Activity: Telomerases are necessary to sustain cell divisionunrestricted by the Hayflick limit of ˜50 cell divisions. hESCs expresshigh telomerase activity to sustain self-renewal and proliferation, andiPSCs also demonstrate high telomerase activity and express hTERT (humantelomerase reverse transcriptase), a necessary component in thetelomerase protein complex.

Pluripotency: iPSCs will be capable of differentiation in a fashionsimilar to ESCs into fully differentiated tissues.

Neural Differentiation: iPSCs could be differentiated into neurons,expressing βIII-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B,and MAP2. The presence of catecholamine-associated enzymes may indicatethat iPSCs, like hESCs, may be differentiable into dopaminergic neurons.Stem cell-associated genes will be downregulated after differentiation.

Cardiac Differentiation: iPSCs could be differentiated intocardiomyocytes that spontaneously begin beating. Cardiomyocytes expresscTnT, MEF2C, MYL2A, MYHCβ, and NKX2.5. Stem cell-associated genes willbe downregulated after differentiation.

Teratoma Formation: iPSCs injected into immunodeficient mice mayspontaneously forme teratomas after certain time, such as nine weeks.Teratomas are tumors of multiple lineages containing tissue derived fromthe three germ layers endoderm, mesoderm and ectoderm; this is unlikeother tumors, which typically are of only one cell type. Teratomaformation is a landmark test for pluripotency.

Embryoid Body: hESCs in culture spontaneously form ball-like embryo-likestructures termed “embryoid bodies,” which consist of a core ofmitotically active and differentiating hESCs and a periphery of fullydifferentiated cells from all three germ layers. iPSCs may also formembryoid bodies and have peripheral differentiated cells.

Blastocyst Injection: hESCs naturally reside within the inner cell mass(embryoblast) of blastocysts, and in the embryoblast, differentiate intothe embryo while the blastocyst's shell (trophoblast) differentiatesinto extraembryonic tissues. The hollow trophoblast is unable to form aliving embryo, and thus it is necessary for the embryonic stem cellswithin the embryoblast to differentiate and form the embryo. iPSCsinjected by micropipette into a trophoblast to generate a blastocysttransferred to recipient females, may result in chimeric living mousepups: mice with iPSC derivatives incorporated all across their bodieswith 10%-90 and chimerism.

ii. Epigenetic Reprogramming

Promoter Demethylation: Methylation is the transfer of a methyl group toa DNA base, typically the transfer of a methyl group to a cytosinemolecule in a CpG site (adjacent cytosine/guanine sequence). Widespreadmethylation of a gene interferes with expression by preventing theactivity of expression proteins or recruiting enzymes that interferewith expression. Thus, methylation of a gene effectively silences it bypreventing transcription. Promoters of pluripotency-associated genes,including Oct4, Rex1, and Nanog, may be demethylated in iPSCs, showingtheir promoter activity and the active promotion and expression ofpluripotency-associated genes in iPSCs.

Histone Demethylation: Histones are compacting proteins that arestructurally localized to DNA sequences that can effect their activitythrough various chromatin-related modifications. H3 histones associatedwith Oct/4, Sox2, and Nanog may be demethylated to activate theexpression of Oct4, Sox2, and Nanog.

IX. Culturing and Differentiation of iPS Cells

After somatic cells are introduced with reprogramming factors using thedisclosed methods, these cells may be cultured in a medium sufficient tomaintain the pluripotency. Culturing of induced pluripotent stem (iPS)cells generated in this invention can use various medium and techniquesdeveloped to culture primate pluripotent stem cells, more specially,embryonic stem cells, as described in U.S. Pat. App. 20070238170 andU.S. Pat. App. 20030211603. It is appreciated that additional methodsfor the culture and maintenance of human pluripotent stem cells, aswould be known to one of skill, may be used with the present invention.

In certain embodiments, undefined conditions may be used; for example,pluripotent cells may be cultured on fibroblast feeder cells or a mediumwhich has been exposed to fibroblast feeder cells in order to maintainthe stem cells in an undifferentiated state. Alternately, pluripotentcells may be cultured and maintained in an essentially undifferentiatedstate using defined, feeder-independent culture system, such as a TeSRmedium (Ludwig et al., 2006; Ludwig et al., 2006). Feeder-independentculture systems and media may be used to culture and maintainpluripotent cells. These approaches allow human embryonic stem cells toremain in an essentially undifferentiated state without the need formouse fibroblast “feeder layers.” As described herein, variousmodifications may be made to these methods in order to reduce costs asdesired.

For example, like human embryonic stem (hES) cells, iPS cells can bemaintained in 80% DMEM (Gibco #10829-018 or #11965-092), 20% definedfetal bovine serum (FBS) not heat inactivated (or human AB serum), 1%non-essential amino acids, 1 mM L-glutamine, and 0.1 mMβ-mercaptoethanol. Alternatively, iPS cells can be maintained inserum-free medium, made with 80% Knock-Out DMEM (Gibco #10829-018), 20%serum replacement (Gibco #10828-028), 1% non-essential amino acids, 1 mML-glutamine, and 0.1 mM β-mercaptoethanol. Just before use, human bFGFmay be added to a final concentration of about 4 ng/mL (WO 99/20741) orzebrafish bFGF may be used instead as in the Examples.

Various matrix components may be used in culturing and maintaining humanpluripotent stem cells. For example, collagen IV, fibronectin, laminin,and vitronectin in combination may be used to coat a culturing surfaceas a means of providing a solid support for pluripotent cell growth, asdescribed in Ludwig et al. (2006a; 2006b), which are incorporated byreference in its entirety.

Matrigel™ may also be used to provide a substrate for cell culture andmaintenance of human pluripotent stem cells. Matrigel™ is a gelatinousprotein mixture secreted by mouse tumor cells and is commerciallyavailable from BD Biosciences (New Jersey, USA). This mixture resemblesthe complex extracellular environment found in many tissues and is usedby cell biologists as a substrate for cell culture.

IPS cells, like ES cells, have characteristic antigens that can beidentified or confirmed by immunohistochemistry or flow cytometry, usingantibodies for SSEA-1, SSEA-3 and SSEA-4 (Developmental StudiesHybridoma Bank, National Institute of Child Health and HumanDevelopment, Bethesda Md.), and TRA-1-60 and TRA-1-81 (Andrews et al.,1987). Pluripotency of embryonic stem cells can be confirmed byinjecting approximately 0.5-10×10⁶ cells into the rear leg muscles of8-12 week old male SCID mice. Teratomas develop that demonstrate atleast one cell type of each of the three germ layers.

Various approaches may be used with the present invention todifferentiate iPS cells into cell lineages including, but not limitedto, hematopoietic cells, myocytes (e.g., cardiomyocytes), neurons,fibroblasts and epidermal cells, and tissues or organs derivedtherefrom. Exemplary methods of hematopoietic differentiation of iPScells may include, for example, methods disclosed by U.S. ApplicationNo. 61/088,054 and No. 61/156,304, both incorporated herein by referencein their entirety, or embryoid body (EB) based methods (Chadwick et al.,2003; Ng et al., 2005). Fibronectin differentiation methods may also beused for blood lineage differentiation, as exemplified in Wang et al.,2007. Exemplary methods of cardiac differentiation of iPS cells mayinclude embryoid body (EB) methods (Zhang, et al., 2009), OP9 stromacell methods (Narazaki, et al., 2008), or growth factor/chemical methods(see U.S. Patent Publn. 20080038820, 20080226558, 20080254003 and20090047739, all incorporated herein by reference in their entirety).

X. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Processing Leukophoresis into Aliquots of PBMCs

The leukophoresis sample (leukopak) was derived from a process in which8 liters of peripheral blood was circulated through a centrifugal fieldin order to concentrate mononuclear cells and limit the amount of redblood cells in a resulting volume of approximately 125 ml. The leukopakwas further processed as follows: One end of a leukopak bag was swabbedwith alcohol swab and cut with razor blade to drain into a flask. Thevolume was diluted to between approximately 500 ml with Hank's solutionand then aliquoted into 16-29 tubes with 50 ml capacity, 30 ml per tube.The tubes were spun at 400 g for 30 minutes with no brake and noacceleration. White liquid was aspirated and new 50 ml tubes were filledup halfway and topped off with 25 ml PBS. This procedure was repeated 2additional times for a total of 3 washes. Cells were counted before thelast wash using a hemacytometer. The yield was between 30-60 tubes of1×10⁸ cells/tube.

To process whole blood the blood draws were collected in a tubecontaining an anticoagulant or a CPT tube. A brief outline forprocessing blood samples obtained by the CPT tube is provided, Brieflytransfer approximately 7-8 ml from the upper (plasma) phase into a 50 mlsterile tube. Dilute to 50 ml with calcium-free PBS. Invert to mix.Centrifuge for 15 minutes at 300 RCF. Remove approximately 95% of thesupernatant without disturbing the pellet. Transfer the supernatant intoa separate 50 ml tube. Gently resuspend the pellet by tapping the tube.Add 20 ml of calcium-free PBS. Invert to mix. Transfer half(approximately 10 ml) to a 15 ml tube. Centrifuge both tubes for 15minutes at 300 RCF. Remove as much of the supernatant as possiblewithout disturbing the pellet. Resuspend the pellet in the 50 ml tubewith 2 ml of medium suitable for T cells (AIM-V based Medium) or CD34medium (Stem Pro based medium) as described below and take a viable cellcount using the cedex cell counter.

If the blood sample is collected in tubes containing an anticoagulantlike EDTA the processing step involves the lysis of red blood cellsfollowed by a Ficoll gradient separation of PBMCs from the blood sample,The sample is initially diluted with equal volume of calcium-magnesiumfree PBS. The red blood cells are lyzed using the ACK buffer(Invitrogen) according to the manufacturer's' instructions. The cellsuspension without the red cells is washed and layered on a Ficollgradient as described before for the leukopak samples. The PBMCs areobtained from the buffy coat are washed again using calcium-magnesiumfree PBS and resuspended medium suitable for T cells (AIM-V basedMedium) or CD34 medium (Stem Pro based medium).

EXAMPLE 2 T-Cell Activation and Expansion

Peripheral Blood Mononuclear Cells (PBMCs) were obtained from BiologicalSpecialty Corp (Colmar, Pa.) donor #33231 (“Donor A”). A leukocyte packwas processed with Lymphocyte Separation Medium (Cellgro) to yield PBMCsas described above, which were in turn frozen into aliquots and storedin liquid Nitrogen. Aliquots were thawed and expanded in freshlyprepared AIM-V Medium+pen/strep/glutamine (AIV-V/ps/s/g media)(Invitrogen) plus 300 IU/ml rhIL2 (Peprotech) and 10 ng/ml solubleanti-CD3 antibody (OKT3 clone, eBiosciences). Several days afteractivation exponential growth was verified by CEDEX cell count. After 3days in culture cells were assayed for T-cell phenotype and thentransduced with the reprogramming factors. In one experiment T-cellphenotype was not verified before plating or after transduction. ThisT-cell activation experiment was repeated multiple times andconsistently provided the same result—T-cells made up 90% or more of theculture post activation and post transduction. It was confirmed that itwas the T-cells that were transduced with the reprogramming factor(s).

Details of the T-cell activation and expansion procedure (Table 1). PBMCvials were thawed to collect 75×10⁶ cells (approximately 3 vials). Thecontents of each PBMC vial were added to 7 ml AIM-V/p/s/g media. Thecell suspension was centrifuged at 1200 rpm for 4 min. The pellet wasresuspended in 10 ml AIM-V+p/s/g. The cell concentration was adjusted to1×10⁶ cells/ml in a total of 28 ml and 2×10⁶ cells/ml in a total of 25ml. IL2 (300 IU/ml) and OKT3 (10 ng/ml) were added to cell suspensionsand mixed. The cells from each concentration were plated at 1.5 ml perwell in a 24-well tissue culture plate and incubated at 37° C. A totalof 18 wells of 1×10⁶ cells/ml (1.5 ml/well) and 16 wells of 2×10⁶cells/ml (1.5 ml/well) were used. Cell counts were verified and recordedas day 0. Day 0 cell counts were compared with day 3 and day 4 counts toverify exponential expansion.

TABLE 1 T-cell Activation and Expansion Cell PBMC (T-cell) Number MediaCytokines Antibody 24 well plate 1-2 × 10⁶ AIM-V + 1X 300 IU/ml 10 ng/mlOKT3 cells/well pen/strep/ IL2 (anti-CD3) - glutamine make fresh 10 ng/ul tube from 1 ug/ul stock in PBS−/−

EXAMPLE 3 Retrovirus Production

Retroviral vectors Nanog RFP, Lin28 RFP, Oct4 eGFP, and Sox2 eGFP wereconstructed as described previously (see U.S. Application No.61/088,054, incorporated herein by reference). Retroviral vectors c-MycRFP, Klf4 RFP, Oct4 eGFP, and Sox2 eGFP were constructed similarly. Tocounteract the possible toxic effects of c-Myc expression, retroviralvector SV40 large T gene (SV40LT)-RFP may be constructed and used insome of the combinations (Yu et al., 2009).

Details of 293T cell preparation procedure (Table 2): cells were seededapproximately 24 hours prior to transfection. The number of cellsnecessary to yield adequate volumes of viral supernatant for theexperiment being performed was calculated. Media were aspirated and 293Tplates were washed with 5 ml PBS and then aspirated. 1 ml of 0.05%Trypsin/EDTA per 10 cm plate was added and distributed evenly. Theplates were incubated at room temperature for 2-5 minutes, firmly tappedagainst hand or wall of hood to dislodge cells, and added 4 ml of D10F.293T cells were triturated (pipet 3-4 times) to ensure single cellsuspension and transferred to 15 ml conical tube. 300 ul of 293T cellswere removed for counting on CEDEX cell counter. Cell concentration wasadjusted to 5×10⁵ cells/ml in D10F media. Ten ml of cell suspension wasplated for each 10 cm plate needed for the experiment (5×10⁶ cells perplate).

TABLE 2 293T Cell Preparation Cell Viral Supernatant 293T Number YieldMedia 10 cm plate 5 × 10⁶ 5 ml D10F

Transient Transfection for Retrovirus Production: 293T cells were seededat 5×10⁶ cells per 10 cm dish and incubated overnight. The next day thecells were transfected with 10 ug of MMLV retroviral vector, 3 ug ofGag/pol, 1 ug of NFkB, and 1 ug of VSVg using PEI (Sigma) lipophilicreagent and OptiMEM (Invitrogen). 500 ul of OptiMEM was incubated with40 ul of PEI for 5 minutes. In a separate tube, 10 ug of retroviralvector+3 ug of Gag/pol+1 ug of NFkB+1 ug of VSVg were added to 500 ul ofOptiMEM. PEI/OptiMEM mixture was added to DNA/OptiMEM mixture for atotal of approximately 1 ml and incubated for 25 minutes. During theincubation, recipient 293T plates were washed with 10 ml PBS−/− and 4 mlDMEM without FBS was added. The DNA/PEI mixture was added drop-wisedirectly onto the 293T cells After four hours, the media was exchangedwith 5 ml of DMEM/10% FBS/50 mM HEPES and incubated. Forty eight hoursafter transfection, fluorescence of 293T cells was visualized to confirmhigh efficiency transfections. The media (5 ml/plate) was collected asvirus containing supernatant. Supernatant was filtered through 0.8 umpore size filter and collected for subsequent transduction.

Details of verifying expansion and phenotype of T-cells (about 1 daybefore reprogramming): T-cells should represent most of the cellpopulation due to the cytokine and antibody addition. Verification wasperformed by surface staining with anti-CD3, anti-CD4 and anti-CD8 flowcytometry antibody. In addition, cell counts were performed. A lag afterthawing was noticed but the cells increased in number from d0; this cellcount was recorded and compared with the count next day to verifydoubling.

EXAMPLE 4 Retroviral Transduction of T-cells (Day 0)

Following 3 days of IL2 and OKT3 activation and expansion, the cellpopulation consisted of 97-99% T-cells. These T-cells were resuspendedat 1e6 cells/well in a volume of 2 ml DMEM (Invitrogen)+10% FBS(Hyclone) with retrovirus containing media, 300 IU/ml rhIL2 (recombinanthuman IL-2) and 4 ug/ml polybrene. Retrovirus containing media wasprepared by transfection of 293T cells with MMLV packaging elements incombination with one of several transcription factors known to beinvolved in reprogramming. After preparing the viruses individually, themedia were combined in two different cocktails and exposed to T-cells;set one included viruses that express the transcription factors Sox2,Oct4, c-Myc, and Klf-4) and set two used viruses that express Sox2,Oct4, Nanog, and Lin28. Separately, cells were exposed to one of the sixviruses to serve as control transductions. The cell culture media wasreplaced with the virus containing media and the cells were subjected tocentrifugation at 1000 g for 1.5 h at 32° C. (spinfection).Subsequently, the cells were incubated for 4 hours at 37° C. Followingincubation, 1 ml of media was carefully aspirated and replaced withfresh DMEM+10% FBS Cells were gently triturated to mix and ensure evenresuspension. Following trituration the cultures were incubated for 18h, timed from the beginning of the spinfection. After 18 h the cellswere harvested, resuspended in fresh viral supernatant+DMEM with 10%FBS+IL2+polybrene, replated in fresh 24 well plates and spinfected asecond time as described above.

Details of the procedure for harvesting retrovirus and transduction ofT-cells (DAY 0):, In addition to the reprogramming factor, eachretrovirus carried a fluorescent protein tag. Thus to confirm a highefficiency of transfection 293T cells were visualized by fluorescencemicroscopy 48 hours after transfection. 293T media (˜5 ml per plate)were collected, centrifuged to remove debris, and the virus-containingsupernatants were filtered through 0.8 um syringe filter and placed inseparate 15 or 50 ml conical tubes. The virus was stored for 0 to 5 daysat 4° C. T-cells were activated and counted on successive days to verifythat they were growing exponentially at the time of infection (on day3). The cells were harvested, centrifuged, resuspended in viruscontaining supernatant, and seeded to 24-well plates at 1e10⁶ cells perwell. The volume used per well for each virus stock is described inTable 3 (total volume=2 ml)). Six separate control transductions werecarried out to verify the infectivity of each individual viral stock.For the latter transductions, 1e10⁶ cells was resuspended in 500 μl ofone of the virus stocks and the total volume was adjusted to 2.0 mlusing D10F and 300 IU/ml IL2+4 ug/ml polybrene. All reprogramming trialswere performed in duplicate or triplicate. Non-transduced cells wereused as a negative control.

TABLE 3 Viral Supernatants for Reprogramming of T-cells ReprogrammingVolume of Gene Viral Set 1 OCT4 500 μl SOX2 500 μl NANOG 500 μl LIN28500 μl or Set 2 OCT4 500 μl SOX2 500 μl C-MYC 500 μl KLF-4 500 μl

Plates were spinfected at 1000 g for 1.5 hours at 32 degrees withacceleration set to ˜4 and brake to ˜4. After spin, plates weretransferred to an incubator to incubate for 4 hours. After 4 hourincubation, plates were carefully transferred to a hood while makingsure not to jostle plates (keep cells settle on bottom of wells). Theplates sat in hood for 5 minutes. One ml of media/virus was carefullyaspirated from the top of the well using a P1000 pipettor. After adding1 ml fresh D10F and 300 IU IL2, the plates were incubated 18 hours at 37degrees. Any unused viral supernatants were stored at 4° C. for secondround of infection.

Details of the procedure for second transduction of T-cells (DAY 1):After 24 hours from initial spinfection start (DAY 0), cells in allwells were collected individually in sterile capped FACS tubes andcentrifuged at 1200 rpm for 4 minutes. Supernatant was aspirated usingfresh 10 ul non-filtered tip on a glass aspirator for each tube/well.Cells were resuspended in appropriate virus(es), IL2, and polybrene asdescribed above. Cells were plated in unused wells of same plate or in anew 24 well plate (wells from first transduction were not reused).Spinfection was followed and steps as described above were repeated.

Details of the procedure for verifying expansion of T-cells (DAY 1): ACedex cell count was performed on left over well of untransduced sample.At this point the cells were increasing exponentially in number from d0.This cell count was recorded and compared with previous day's counts toverify doubling. This well was retained as a negative control as well asfor any further testing. Cells in this well were fed with half-mediaexchanges plus 300 IU IL2/ml as needed.

EXAMPLE 5 Plating Transduced T-cells on MEFs

MEF Plating: MEFs were plated on gelatin coated 6 well plates or 10 cmdishes 1-3 days prior to introducing the transduced cells or iPScolonies (plating MEFs one day prior to transduction may be optimal).

Verification of T-Cell Expansion and Transduction Efficiency: T-cellidentity was verified 2-3 days after activation by flow cytometrysurface staining with anti-CD3, anti-CD4, and anti-CD8, as well aspost-transduction to verify the cell populations that were transducedsuccessfully. CEDEX cell counts were conducted on days 0, 2, 3 and 4 toconfirm exponential expansion and thus amenability to MMLV retroviralinfection.

Plating Transduced T-Cells on MEFs: At day 3 post initial transductionsuccess and efficiency estimates were verified by fluorescent microscopyand flow cytometry as listed above. Transduced cells were added in twocell concentrations (5×10⁶ and 2×10⁶) to 10 cm dish MEF plates in a50:50 media combination of D10F:hES w/o FGF (no added IL2 or othercytokines). Cells were incubated and fed every other day.

Details of the procedure for plating irradiated MEFs: MEFs were plated1-3 days prior to introducing the transduced cells. The number of 10 cmplates needed was calculated (500 k transduced cells per 10 cm plate;transcription factors (set 1 or set 2), untransduced control, c-Myc onlycontrol, MEF only control—5 plates+). 0.1% gelatin was used to coat 10cm plates for at least 1 hour and then aspirated. 15 ml of irradiatedMEF cell suspension (˜7.5×10⁴ cells/ml) was added onto each 10 cm plate.Cells were checked the following day to ensure MEFs had attached.

Details of the procedure for transfer of transduced T-cells toirradiated MEFs (DAY 3): A fluorescent microscope was used to verifytransductions. A Flow cytometer was used to verify transduction anddetermine transduction efficiency. A minimum 20% efficiency isconsidered to be the requirement to proceed with reprogramming (platingonto MEFs, etc). GFP/RFP analysis and surface staining were performed toverify transduction of T-cells was independent from other cellpopulations. 100 ul of cells were collected in FACS tubes and spun at1200 rpm for 4 min. The supernatant was aspirated, and cells wereresuspended in 5 ml FACS Buffer and centrifuged again. The pellet wasresuspended in 150 ul FACS buffer, and stained with anti-CD3, anti-CD4or anti-CD8 flow cytometry antibodies. Cells were analyzed on flowcytometer, to verify CD3⁺ cells (T-cells) were transduced what thetransduced subsets were. Media were aspirated from irradiated MEF platesand 7.5 ml DMEM+10% FBS was added. 5×10⁵ transduced T-cells werecollected, centrifuged at 1200 rpm for 4 minutes, and resuspended in 7.5ml hES medium without bFGF. T-cells were added dropwise to irradiatedMEF plates. No IL2 or other cytokines was added. Then the MEF plateswere incubated at 37 degrees.

EXAMPLE 6 Maintenance and Feeding of MEF-plated Transduced Cells

Days 5-9: Half-media exchanges were performed for each reprogramming 10cm plate with hES media (CM) supplemented with 100 ng/ml of zebrafishFGF. A novel feeding strategy was developed to minimize suspension cellloss while maximizing the positive effects of replenishing of media.Briefly, five 10 cm dish lids were used (and reused for all subsequentfeedings) as props to angle dishes. Dishes were carefully removed fromthe incubator so as to not disturb any loosely adherent cells. Plateswere set on reserved lids at an angle but with no MEFs/cells exposed.Cells were allowed to settle for 10 minutes. After settle period eachlid was removed and 7.5 ml was carefully/slowly aspirated from the verytop of the media horizon on the bottom of the plate. Less than 1% cellloss was verified by collecting this removed media, centrifuging at 1200rpm×4 min, resuspending in 1 ml media and counting on CEDEX. 7.5 ml offresh media was then added dropwise in a circular motion being carefulnot to disturb cells, and dishes were placed back in incubator. Thismethod served the purpose of minimizing cell loss while allowing regularmedia changes. Days 9-30: Half-media exchanges were performed for eachreprogramming 10 cm plate with MEF-conditioned hES media (MEF-CM)supplemented with 100 ng/ml of zebrafish bFGF.

Details of the procedure for maintenance and feeding schedule (DAY5-30): Days 5-9: Half-media exchanges were performed for eachreprogramming 10 cm plate with hES media (CM) supplemented with 100ng/ml of zebrafish FGF. Five 10 cm dish lids were gathered to be usedfor all subsequent feedings as props to angle dishes. 10 cmreprogramming dishes were removed from incubator and set on reservedlids so that plates were at an angle but with no MEFs/cells exposed(media should still be covering the entire surface, pooling at thebottom, and not spilling). Dishes were settled for 10 minutes. Aftersettle period each lid was removed, and 7.5 ml supernatant wascarefully/slowly aspirated from very top of the media horizon on thebottom of the plate. The supernatant or aspirated medium was collected,centrifuged 1200 rpm×4 min, resuspended in 1 ml media and counted onCEDEX. It was verified that less than 1% of the cells were lost. 7.5 mlof fresh media were added dropwise in a circular motion being carefulnot to disturb cells, place back in incubator. Feeding regimen beganevery other day for reprogramming plates. Days 9-30: Half-mediaexchanges were performed for each reprogramming 10 cm plate with MEFconditioned hES Media (MEF-CM) supplemented with 100 ng/ml of zebrafishFGF. Feeding was proceeded with this medium as in Days 5-9.

EXAMPLE 7 Identifying and Picking iPS Colonies

Activated and expanding T cells displayed characteristic cell morphologyand clustering behavior. Detection of retroviral transduction efficiencywas determined by GFP and RFP expression 72 h post initial transduction,over the course of ˜3 weeks the transgenes were silenced and display anhES cell phenotype. Well defined iPS cell colonies began to appear onday 23. GFP and RFP silencing was verified by fluorescent microscopy andcolonies were picked in a dissecting hood using a pipette tip. Colonypieces were then transferred to fresh 6 well plates of irradiated MEFs.The number of colonies were counted to estimate reprogramming efficiencygiven the number of input plated cells. From this point clonal colonieswere fed daily and manually passaged one more time and then expanded asdescribed in detail below.

Details of the Procedure: Morphologically, iPS cell colonies were denseand comprised of small, compact cells with enlarged nuclei and 2distinct nucleoli. Borders of colony were usually defined. iPS colonieshad the GFP and RFP expressed from the integrated viral DNA silenced.Some bona fide colonies lost fluorescence by ˜20 days post transductionand some lost fluorescence after they had been picked and transferred˜35-40 days post-infection. All colonies were lacking GFP and RFPexpression (though some expression was noted in single cells near by) inthe colonies observed here. This may vary among cell type, particularlyas compared to fibroblasts. To pick manually, a pipet tip was drawn in a“tic tac toe board” fashion directly on the colony to break it up into3-6 pieces to increase the probability of freeing stem cells from thesurrounding MEF and T-cells. Picking was avoided until multiple colonieshave formed so as to avoid confounding counts of total colonies, i.e.,if a small chunk of a colony was left, it might resettle and was falselycounted as a new clone. Cells were then transferred directly into arecipient well of a 6 well plate containing MEFs with hES media and 100ng/ml zebrafish bFGF. Proliferation, morphology, and loss offluorescence were then monitored for 1-2 weeks to be confident thatclones were indeed fully reprogrammed. The cells were fed daily afterone day of no feeding following picking. After the picked and platedcolonies adhered and displayed characteristic ES-like morphology, theseES-like colonies were manually picked as described above again onto anew set of 6 well irradiated MEF plates and fed daily. As wells becameconfluent, the cells were passaged as normal iPS cell lines with 1 mg/mlcollagenase (Yu, et. al., 2007). iPS cells were frozen down at variouspassages, and test thaws performed on each set.

Clonal iPS colonies formed d23−30=21 colonies (all from set 1 factors asof d30), 7 from high transduced-cell seeding density (2×10⁶ per 10 cmdish) and 14 from low density (5×10⁵ per 10 cm dish). Dishes were feduntil it was determined that no additional colonies would grow out. Atotal of iPS lines were obtained, frozen and expanded.

EXAMPLE 8 Derivation of Induced Pluripotent Stem Cells from HumanPeripheral Blood T Lymphocytes

Activated T-cell enriched populations containing 1×10⁶ cells weresubjected to two rounds (at day 0 and 1) of retroviral transduction withfour separate vectors, each encoding one of the reprogramming factors(SOX2, OCT4, c-Myc, or KLF4) linked to a fluorescent marker gene (arepresentative vector map is shown in FIG. 10). Transduction efficiencywas assessed at day 3 by fluorescence microscopy and flow cytometry.Staining for CD3 showed the transduced population to be 99%+/−1% CD3⁺(FIG. 2A).

T-cells are well suited as a starting material for reprogramming due totheir abundance in whole blood (˜6.5×10⁵-3.1×10⁶/ml in healthy adults)(Lichtman and Williams, 2006) and ease of culture using well-establishedprotocols (Johnson et al., 2009; Morgan et al., 2006). To facilitateT-cell proliferation and efficient retroviral transduction, peripheralblood mononuclear cells (PBMCs) were isolated from a leukapheresis or astandard venipuncture (Vacutainer© CPT tube) to be reprogrammed into iPScells (FIG. 1). PBMCs from a non-mobilized donor were activated withanti-CD3 antibody and expanded in the presence of IL-2 in serum-freemedia (FIG. 2A). This led to preferential expansion of mature CD3⁺T-cells consisting of an average day 3 CD3⁺ purity of 90%+/−7% (FIG.2A).

The population which was skewed predominantly towards T cells was thentransduced with the reprogramming factors. The population of cellscontaining the transduced T-cells was placed on irradiated mouseembryonic fibroblasts (MEFs) in hESC medium supplemented with 100 ng/mlbasic fibroblast growth factor (bFGF). iPSC colonies were observedbeginning at day 23. Reprogramming efficiencies of T-cells wereestimated by dividing the number of colonies with hESC-like morphologyby the input number of transduced cells and determined to beapproximately 0.01%, similar to published fibroblast and CD34⁺ cellreprogramming efficiencies (Yu et al., 2007; Loh et al., 2009).

TiPS were generated from both leukapheresis samples (from a maleHispanic adult, lines denoted “TiPS-L”) and whole blood Vacutainer©samples (from a male Caucasian adult, lines denoted “TiPS-V”). In eachcase, reprogramming was achieved using an input cell number equivalentto the amount of T-cells in 1 ml whole blood. Colonies displaying hESCmorphology were expanded on MEFs and the clones were successfullymaintained under feeder-free conditions using mTeSR media and Matrigelcoated plates.

Pluripotency was verified by expression of hESC pluripotency markersSSEA-3, SSEA-4, Tra-1-81, and OCT4 using flow cytometry (FIG. 2B) andalkaline phosphatase staining (FIG. 8).

DNA fingerprinting was also performed to verify that TiPS shared agenetic background with the starting donor T-cell population and to ruleout cell line cross-contamination (FIG. 7). STR (short tandem repeats)analysis showed that the iPS colonies were derived from the donor'sgenetic material. The donor PBMC and the iPS line were male genderspecific and are identical to each other for 15 allelic polymorphismsacross the 8 STR loci analyzed (Table 4, below).

TABLE 4 Confirmation of Cell Identity via Polymorphisms PBMC LocusRepeat # TiPS1ee Donor A Fib-iPS D16S539 5, 8-15 11, 12 11, 12 10, 13D7S820 6-14  8, 10  8, 10  9, 12 D13S317 7-15  8, 12  8, 12 11, 13D5S818 7-15 12, 13 12, 13 12, 13 CSF1PO 6-15 12, 12 12, 12 11, 13 TPOX6-13  9, 11  9, 11 8, 9 Amelogenin NA X, Y X, Y X, X TH01 5-11 7, 9 7, 9  8, 9.3 vWA 11, 13-21 16, 18 16, 18 16, 19

The TiPS lines' T-cell origin via multiplex PCR detection of TCR β chainrearrangements was confirmed (FIG. 2C). T cells have a single productiveV-J rearrangement in the TCR beta chain and should retain thischaracteristic gene sequence after becoming TiPS cells; using a mastermix combining various primers for the most common beta chainrearrangements PCR amplification showed one band of unique size andsequence as determined by fragment analysis electropherogram on an ABI3730 DNA analyzer. iPS cells derived from fibroblasts, “Fib-iPS” wereused as a negative control.

TiPS clones expressed human embryonic stem cell marker genes DNMT38,LEFTS, NODAL, REX1, ESG1, TERT, GDF3, and UTF1 (FIG. 3A). Total RNA wasisolated from H1 hES cells, Fib-iPS (derived from fibroblasts), T-cellsfrom the primary donor, and TiPS clones TiPS1ee and TiPS1b were analyzedusing RT-PCR. Further characterization demonstrated integration of thetransgenes into the host genome as well as their silencing followingsuccessful reprogramming (FIGS. 3B-3C). TiPS were similar to both thehESC line H1 and to fibroblast-derived iPSC line controls in all of theabove assays. Endogenous and exogenous (transgene) expression ofreprogramming genes showed complete reprogramming as evidenced bysilencing of transgene expression (FIG. 3C). GAPDH was used asamplification control in both A+B. Genomoic DNA was isolated andanalyzed by PCR to confirm integration of reprogramming genes by usingforward primers for the gene of interest and reverse primers for theIRES (FIG. 3B). OCT4 forward and reverse primers were used as a PCRreaction control.

TiPS clones expressed human embryonic stem cell-specific pluripotencymarkers as shown by flow cytometry analysis (FIG. 3D), AlkalinePhosphatase staining, and karyotipic analysis by Gbanding chromosomeanalysis. Lines were karyotypically normal after multiple passages andhave been propagated for over 30 passages in culture while retaining anormal karyotype (FIG. 9).

Finally, the TiPS cell lines were evaluated to determine their in vivoand in vitro differentiation potential. TiPS clones formed teratomascontaining tissue consistent with derivation from all three primary germlayers (FIG. 4A). The cell lines were also assessed for their capabilityto differentiate in vitro into ectodermal and mesodermal lineages invarious directed differentiation protocols. The clones were able togenerate neurons, beating cardiac troponin T-positive cardiomyocytes andmultipotent granulocyte-erythroid-macrophage-megakaryocyte (GEMM)hematopoietic cells (FIGS. 4B-4E).

TiPS were differentiated into multiple cell types. TiPS weredifferentiated into cardiomyocytes by the following method. TiPS clonesformed embryoid bodies (EBs) and were differentiated into cardiomyocytesvia HGF/bFGF mediated cardiac induction (FIG. 4C). Beating cardiomyocyteaggregates were observed on day 14. TiPS were also differentiated intoblood (FIG. 4E). Hematopoietic progenitor cells (HPCs) were derived fromEBs using a combination of BMP-4, VEGF, Flt-3 ligand, IL-3, GM-CSF andFGF-2. Functional capability of TiPS1ee-derived HPCs was determinedusing the colony-forming unit (CFU) assay. CFU-GM, BFU-E, and CFU-GEMMcolonies were observed at day 12.

In summary, iPS cells were successfully generated from T cells derivedfrom the peripheral blood of a non-mobilized donor. The amount ofstarting material was adaptable to 1 ml of starting material from astandard vacutainer. TiPS reflected the identity of the host material.TiPS also harbored hallmark characteristics of normal human ES cells andiPS cells derived from other cell sources. TiPS were furtherdifferentiated into multiple cell types, including beating cardiomyocyteaggregates and blood cells.

No significant differences in differentiation potential between TiPSclones and hESC lines or fibroblast-derived iPSC lines were observed(FIGS. 4D-4E). The potential effect of the persistence of TCR generearrangements in the iPSC genome on subsequent differentiation may betested.

TCR rearrangements may in fact prove advantageous in certain contexts,such as for iPSC clone tracking, as demonstrated by the detection ofparent line clonal TCR β chain rearrangements in derivative teratomas.(FIG. 5). Further, upon re-differentiation into T-cells TiPS cells maybypass key steps in the canonical thymic development sequence due to themechanism of TCR allelic exclusion caused by the expression of theirpre-rearranged TCR genes. This phenomenon could be explored in T-celldevelopment studies.

It should be noted that insertional mutagenesis and other potentialdisruptions of cellular function are possible when using a retroviralreprogramming protocol (Mirxhwll et al., 2004). Recent advances in usingepisomal reprogramming methods may address these issues and efforts arein progress to reprogram T-cells via these alternative methods (Yu etal., 2009; Zhou et al., 2009). Further, an interesting example of apotential therapeutic use for such episomally reprogrammed TiPS cells isas a source to differentiate integration-free hematopoietic stem cellsbearing endogenous TCR genes specific for tumor-associated antigens (vanLent et al., 2007).

Previous reports of reprogramming terminally differentiated Blymphocytes in mice required the addition, or knock-down, of cellularidentity-associated transcription factors and used adoxycycline-inducible expression system (Hanna et al., 2008). Recently,a description of reprogramming murine T-cells was publishednecessitating a p53 gene knock-out for successful iPSC generation (Honget al., 2009). Experiments involving manipulation of anti-proliferativepathways (Li et al., 2009; Marion et al., 2009; Kawamura et al., 2009;Utikal et al., 2009) offer insights into the mechanisms of reprogrammingand may significantly augment reprogramming efficiencies. However, noneof the above mentioned manipulations appear to be a requirement forsuccessful viral reprogramming of human T-cells. Additionally, our data,coupled with methodologies used in reprogramming adult CD34⁺hematopoietic progenitor cells (Loh et al., 2009; Ye et al., 2009), nowafford a primary, human system to examine recent observations in themouse system correlating differentiation stage of input cells withreprogramming efficiency (Eminli et al., 2009).

The derivation of iPSCs from small, clinically advantageous volumes ofnon-mobilized human peripheral blood was discovered. T-cells representan abundant cell source for reprogramming which can be harvested fromlarge numbers of donors in a minimally invasive manner and cultured viawell-established protocols. In the experiments TiPS were found to havesimilar characteristics and differentiation potential as hESC lines andfibroblast-derived iPSC lines. Additionally, TiPS provide a novel modelwith which to explore iPSC clone tracking, T-cell development andtherapeutic applications of iPSC technology.

Materials and Methods

Cell Growth Media and Basic Fibroblast Growth Factor—iPSC lines weremaintained using previously described methods (Yu et al., 2007).Zebrafish bFGF was substituted for human bFGF in all experiments, aspreviously described (Ludwig et al., 2006a).

Fibroblast iPSC Lines— Control fibroblast-derived iPSC lines, referredto as “Fib-iPS”, were produced as previously described using IMR90 cellsobtained from ATCC (Manassas, Va.) (Yu et al., 2007).

T-cell Activation and Expansion—Peripheral Blood Mononuclear Cells(PBMCs) were obtained from an HLA-A2 positive male Hispanic adult donor(“Donor L”) leukocyte pack (Biological Specialty Corp, Colmar, Pa.)processed with Lymphocyte Separation Medium (Cellgro, Manassas, Va.).Additionally, whole blood samples were collected from a male Caucasiandonor of unknown serotype (“Donor V”) via standard venipuncture in aVacutainer© CPT™ tube (BD Biosciences, San Jose, Calif.) and PBMCscollected by centrifugation according to the manufacturer'srecommendations. Blood samples were obtained with written informedconsent in accordance with the Declaration of Helsinki and InstitutionalReview Board approval from the Biological Specialty Corporation (Colmar,Pa., USA). T-cells were expanded in freshly prepared AIM-V Medium(Invitrogen, Carlsbad, Calif.) supplemented with pen/strep/glutamine(Invitrogen) plus 300 IU/ml rhIL2 (Peprotech, Rocky Hill, N.J.) and 10ng/ml soluble anti-CD3 antibody (eBioscience, OKT3 clone, San Diego,Calif.) (Chatenoud, 2005; Berger et al., 2003). Proliferation wasverified by CEDEX (Roche Innovatis, Bielefeld, Germany) cell count after3 days in culture at which point cells were assayed for T-cell phenotypeand then transduced with reprogramming factors.

Transient Transfection for Retrovirus Production—Retrovirus wasgenerated by transfecting 293T cells in a 10 cm plate at 70-80%confluence with 10 ug of retroviral vector (Moloney Murine LeukemiaVirus) backbone encoding each of 4 reprogramming genes and a fluorescentmarker gene (GFP or RFP), 3 ug of Gag-Pol, 1 ug of plasmid encoding aderivative of NFkB, and 1 ug of Vesicular Stomatitis Virus G proteinusing polyethylene imine (“PEI”) lipophilic reagent (40 ug/10 cm plate).After four hours, the medium was exchanged with 5 ml of DMEM(Invitrogen) plus 10% FBS (Hyclone, Waltham, Mass.) and 50 mM HEPES(Invitrogen). Viral supernatant was collected 48 hourspost-transfection, centrifuged, and passed through a 0.8 um pore sizefilter.

Retroviral Transductions Via Spinfection—One million activated donorcells per well were “spinfected” via centrifugation for 1.5 h×1000 g at32° C. in a mixture of the four retroviral supernatants plus 4 ug/mlpolybrene (Sigma-Aldrich, St. Louis, Mo.), and 300 IU/ml rhIL-2. Afterspinfection the plates received a half-media exchange, and wereincubated overnight. The next day the cells were harvested bycentrifugation and spinfected a second time.

Verification of T-Cell Expansion and Transduction Efficiency—T-cellidentity was verified 3 days after activation by flow cytometry surfacestaining with anti-CD3 antibodies (BD, clone HIT3a), as well aspost-transduction to verify which cell population was transducedsuccessfully. Samples were run on an Accuri (Ann Arbor, Mich.) flowcytometer. CEDEX cell counts were conducted on days 0, 3 and 4 toconfirm expansion and thus amenability to MMLV retroviral infection(data not shown).

Plating Transduced T-Cells on MEFs—Seventy two hours post initialtransduction, transduction success and efficiency estimates wereverified by fluorescent microscopy and flow cytometry as listed above.5×10⁵ transduced cells were added to 10 cm plate seeded with MEFs 1 to 3days prior in a 50/50 media combination D10F:hESC without zbFGF (oradditional cytokines). Cells were incubated and fed hESC media+100 ng/mlzbFGF (first week) or MEF-conditioned media+100 ng/ml zbFGF (thereafter)by half media exchange every other day. To avoid cell loss duringfeedings the plates were angled slightly for 10 minutes to allow thecells to settle and media was removed slowly from the media horizon.

iPSC Colony Identification and Picking—Colonies with well-definedborders and typical hESC morphology began to appear around day 23. GFPand RFP silencing was verified by fluorescent microscopy and the numberof colonies were counted to estimate reprogramming efficiency given thenumber of input plated cells. Colonies were manually harvested,transferred to MEFs, and expanded according to established protocols(Maherali and Hochedlinger, 2008; Thomson et al., 1998). Estimates ofreprogramming efficiency were obtained by dividing total number ofputative iPSC colonies by the input number of transduced cells. Countswere ceased after colony harvest (day 25-30) to avoid the inclusion offalse positive re-seeded colonies left behind from the harvest.

DNA Fingerprinting—TiPS cell lines and donor PBMCs were sent to theUniversity of Wisconsin Histocompatibility/Molecular DiagnosticsLaboratory (Madison, Wis.) for short tandem repeat (STR) analysis.Genotypes for 8 STR loci were determined from TiPS cell sample DNA.

Karyotyping—G banding analysis was conducted by WiCell ResearchInstitute (Madison, Wis.).

T-Cell Receptor β Chain Rearrangement Analysis—Genomic DNA was isolatedper manufacturer's protocol (using the Qiagen DNeasy Blood and Tissuekit) from donor T-cells, the TiPS cell lines, and a fibroblast(non-T-cell) derived iPSC line used as a negative control. Additionally,DNA was isolated from frozen teratoma samples and parent cell lines byfirst dissolving tissue and cell samples in a buffer containing Tris,NaCl, EDTA, SDS and Proteinase K (Invitrogen). DNA was then precipitatedwith saturated NaCl and ethanol, and resuspended in water for PCRanalysis. PCR was performed using a multiplex primer kit (InvivoscribeTechnologies, San Diego, Calif.) specific for a majority of clonal TCR βchain rearrangements (van Dongen et al., 2003). Capillaryelectrophoresis and PCR product fragment analysis was performed at theUniversity of Wisconsin Biotechnology Center DNA Sequencing CoreFacility (Madison, Wis.) using an ABI 3730 DNA analyzer. Data wasanalyzed using Peak Scanner software (ABI, Foster City, Calif.).

Alkaline Phosphatase (AP) Staining—Confluent cells grown on MEFs were APstained with Vector Blue Alkaline Phosphatase Substrate Kit III (VectorLaboratories, SK-5300, Burlingame, Calif.) according to themanufacturer's protocol.

RT-PCR for Transgene and hESC Marker Gene Expression—Total RNA wasisolated using the RNeasy Mini Kit (Qiagen, Germantown, Md.) accordingto the manufacturer's protocol. First strand cDNA synthesis was carriedout with oligo-dT primers (as described previously (Yu et al., 2009;Takahashi et al., 2007)) using SuperScript III First Strand Synthesiskit (Invitrogen) according to the product protocol. cDNA was diluted 1:2and PCR reactions were performed with GoTaq Green Master Mix (Promega,Madison, Wis.) using a Mastercyler (Eppendorf, Hauppauge, N.Y.).

PCR Analysis of Viral Integration—Genomic DNA was isolated from 1-5×10⁶iPSCs using DNeasy Blood and Tissue kit (Qiagen) according to themanufacturer's protocol for cultured cells. Genomic DNA (5 ul) was usedfor PCR reactions to check for viral integration using GoTaq GreenMaster Mix (Promega). Specific primer sets were used that detect onlythe transgene and not the endogenous gene. Primers for endogenous OCT4served as a positive control for the reaction. Reactions were performedwith primers as described previously (Yu et al., 2009; Takahashi et al.,2007).

Flow Cytometry: iPSC Line Intracellular and Surface Pluripotency MarkerCharacterization—TiPS maintained on Matrigel were harvested and stainedfor the presence of Tra-1-81 (BD Pharmingen or Stemgent, San Diego,Calif., both clone Tra-1-81), SSEA-3 (BD Pharmingen, clone MC631) andSSEA-4 (BD Pharmingen, clone MC813-70). Intracellular OCT4 (BD, clone40/Oct-3) staining was performed on cells fixed with 2% paraformaldehydeand permeabalized with PBS+0.1% saponin. Cells were stained overnightand analyzed the next day on an Accuri flow cytometer.

Hematopoietic Differentiation and Colony-Forming UnitAssays—Undifferentiated TiPS were adapted to feeder-free conditions onMatrigel coated plates and maintained using mTeSR medium (Stem CellTechnologies, Vancouver BC, Canada). The colonies were harvested usingTrypLE (Invitrogen) and placed in serum-free embryoid body (EB) basalmedia [containing IMDM, NEAA, Glutamine (Invitrogen) and 20% BIT-9500(Stem Cell Technologies) and ROCK inhibitor H1152 in low-attachmentplates to facilitate aggregate formation. Following aggregate formation,the cells were placed in EB basal media supplemented with growth factorsand cytokines: rhBMP-4 (R&D Systems, Minneapolis, Minn.), rhVEGF, zbFGF,rhFlt-3 ligand, rhIL-3, and rhGM-CSF (Invitrogen) for 12 days. The cellswere harvested and the phenotype generated by each iPSC clone wasassessed by surface staining for CD31, CD34, CD43, CD45, CD41 and CD235aby flow cytometry. The individualized cells were placed in MethoCult(Stem Cell Technologies) media for assaying colony-forming units per themanufacturer's instructions.

Assay for Teratoma Formation—Characterized iPSCs cultured on MEFs wereinjected intramuscularly into the hind limb of SCID/beige mice (HarlanLaboratories, Madison, Wis.). Three mice were injected per cell line,each with one 6-well plate of cells. Matrigel (BD Biosciences) was addedat ⅓ total volume to the cell suspension prior to injection. Tumorsformed at 5 to 12 weeks and were processed for hematoxylin and eosinstaining and histological analysis by the McArdle Laboratory for CancerResearch (University of WI-Madison). All animal work was conductedaccording to relevant national and international guidelines under theapproval of the Cellular Dynamics International Animal Care and UseCommittee.

Cardiac Differentiation—Cardiogenesis was induced via a cell aggregatemethod. Briefly, TiPS cells grown on MEFs were harvested withCollagenase IV (Invitrogen) and cells grown on Matrigel were dissociatedinto single cell suspension using Sodium Citrate. The cell suspensionwas allowed to form aggregates in ultra-low attachment flasks in thepresence of recombinant human hepatocyte growth factor (HGF) and/orzbFGF. Additionally, ROCK inhibitor H1152 was added to Matrigel-sourcedcell suspensions. Beating aggregates were dissociated and stained forCardiac Troponin T (cTnT) (Abcam, Cambridge, Mass., clone 1C11) on day14 to 15.

Neuronal Differentiation—The neural differentiation of TiPS cells wasperformed as previously described (Ebert et al., 2009). Briefly, TiPSgrown on MEFs were partially dissociated with Collagenase IV andcultured in suspension as aggregates in Stemline Neural Stem CellExpansion Medium (Sigma-Aldrich) supplemented with B27 supplement(Invitrogen), bFGF (100 ng/ml) and epidermal growth factor (100 ng/ml,Chemicon, Billerica, Mass.). Cultures were passaged weekly using aMcIIwain tissue chopper. To induce neural differentiation, spheres weregrown in neural induction medium (DMEM/F12 plus N2 supplement,Invitrogen) for one week and then plated onto poly-ornithine/laminin(Sigma-Aldrich)-coated coverslips in the same neural induction mediumsupplemented with cAMP (1 uM, Sigma-Aldrich), ascorbic acid (200 ng/ml,Sigma-Aldrich), brain-derived neurotrophic factor and glial cellline-derived neurotrophic factor (both 10 ng/ml, R&D Systems) for afurther weeks. The expression of neuronal maker beta III-tubulin wasanalyzed by immunofluorescence staining as previously described (Zhanget al., 2001).

EXAMPLE 9 Retroviral Reprogramming of T-cells from Cryopreserved HumanPeripheral Blood Patient Samples

This Examples presents the protocol used in the “10 donor” experiment.In that experiment, reprogramming was done as a trial on ten patientsamples and each of the ten patient samples successfully reprogrammed.As shown in FIGS. 6A-6B, Tra-1-60 staining of IPS colonies on MEFs in a96-well format with low number of input T cells. This demonstrates theefficiency of the T-cell approach.

This Example describes a set of procedures (Procedures 1-11 in detailbelow) for efficient retroviral reprogramming of human peripheral bloodT-lymphocytes, particularly the multiple steps and the timing necessaryto achieve Moloney murine leukemia virus (MMLV)-based reprogramming ofhuman peripheral blood T-lymphocytes. The Example focuses on the use ofcryopreserved cells and freshly prepared virus supernatants thatcomprise dual-gene MMLV vectors Oct4-Sox2 and c-Myc-Klf4, orNanog-Lin28. The procedures in the Example may be adapted for use withother vector systems and may also be used for non-cryopreserved sample.

1. Preparatory Procedures:

Prior to ordering and/or receiving peripheral blood samples, establishand maintain an actively growing culture of adherent 293T cells and aseparate culture of non-adherent Jurkat cells. 293T cells are propagatedto meet the demand for virus production. Virus production requires theuse of several vectors and helper plasmids described in “Prepare MMLVreprogramming virus vectors” below. It is necessary to prepare these DNAsamples before proceeding to this step. Finally, it is also recommendedthat an excess supply of MEF-conditioned media is prepared prior to“Prepare MMLV reprogramming virus vectors.”

2. Prepare and Cryopreserve Peripheral Blood Mononuclear Cells (PBMCs):

The following describes a procedure for isolating human peripheral bloodmononuclear cells (PBMCs) from Vacutainers® CPT™ tubes of humanperipheral blood and cryopreservation of PBMCs. The procedure isintended to facilitate derivation of iPS cells, Blood was drawn into aseparate (SST) tube and the tube was sent to an appropriate servicelaboratory for infectious disease testing. The blood sample wascollected in CPT Vacutainer© and sent to the inventors. Upon receipt ofthe samples, they were stored at 4° C. in the proper biocontainmentdevice. The donor information was recorded in a database and anidentifying letter or number was assigned to this donor. The receipt ofinfection disease testing data that demonstrates negativity was alsodocumented as defined by a Safety Committee.

After receipt of blood samples, PBMCs were isolated from CPT Vacutainer©by Sorvall Legend RT centrifuge (using biocontainment adapters ifavailable) at 600×g for 25 minutes at 4° C. and the pellet wasresuspended in 10 ml of cold PBS (for cryopreservation) or RPMI+P/S (forlive cell culture). Cells were counted by using a Cedex instrument.Alternatively, perform replicate counts using trypan blue and ahemacytometer. Count the samples and record the number of viable cellsper ml, and also the percent viability. Centrifuge at 400×g for 15minutes at 4° C. and aspirate the supernatant to eliminate residualclotting factors.

After isolation, PBMCs were prepared for cryopreservation byresuspending the pellet in cold CryoStor10 at approx 10×10⁶ cells/ml andtransferring to pre-cooled cryovials. Typically the yield from one 8 mlCPT Vacutainer© is 15-20 million cells and is divided to two cryovials.Place the cryovials in a pre-cooled Mr. Frosty canister, then transferthe canister into a −80° C. freezer overnight. The following day,transfer the cryovials to a liquid nitrogen storage tank for long termstorage.

3. Prepare MMLV Reprogramming Virus Vectors:

To maintain optimal virus activity, it is recommended that the virussupernatant is stored at 4° C. for less than 4 days prior to use. Thisprotocol describes the production of retrovirus-containing media bytransient transfection of MMLV-based reprogramming bicistronic vectorsOct4-Sox2, cMyc-Klf4, and Nanog-Lin28 (vector maps are represented inFIGS. 11A-11C). It is intended to facilitate derivation of iPS cells byretroviral transduction of human T-cells in 96-well format using acombination of two or three of these vectors.

Propagate and Expand 293T Cells Over the Course of Several Days (orWeeks). The extent of the scale up will depend upon the number of cellsneeded for the transient transfection method described below, and thecorresponding volumes of virus containing supernatant generated.Formulas are provided below to calculate these values.

Preparation for Virus Production. MMLV reprogramming vectors aredesignated Oct4-Sox2, cMyc-Klf4, or Nanog-Lin28 corresponding to thenames of the vector plasmids (as represented in FIGS. 11A-11C), andreferred to here as OS, CK, and NL, respectively. Reprogramming may beachieved through the use of OS+CK, OS+NL, or a combination of all threevectors (OS+CK+NL). An excess of each vector plasmid DNA, as well as thehelper plasmids described below, must be prepared prior to initiatingthis protocol. It is also recommended that a control MMLV plasmid(Sox2-GFP) be prepared.

Determine the number of wells (n) containing target cells that willreceive virus, and which combination of viruses each well will receive.For example, ten (10) different donor T-cell samples were seeded to 7wells each in 96-well format and activated as described below; theyoccupy a total of 70 wells: Two wells from each donor will receive OS+CKreprogramming viruses (nOS+CK=20); Two wells will receive OS+NLreprogramming viruses (nOS+NL=20); Two wells will receive a controlSox2-GFP virus (nGFP=20); The one remaining well will represent anon-transduced control.

Calculate the volume of supernatant media (V) from each vector requiredby use the following equation: V=(n)×(dose)×F; where dose=ml of virusapplied to each well (typically 0.05 ml), and F represents theconcentration factor (typically 50-fold) achieved after precipitation ofthe supernatant (in the step Concentrating the virus below). Followingthe example above, the total number of wells receiving the OS virusn_(OS+CK)±n_(OS+NL)=40. Assuming dose=0.05 ml, and F=50, calculateV_(OS)=(n_(OS+CK)+n_(OS+NL))×(dose)×F=40×(0.05)×50=100 ml. CalculateV_(CK)=(n_(OS+CK))×(dose)×F=20×(0.05)×50=50 ml. CalculateV_(NL)=(n_(OS+NL))×(dose)×F=20×(0.05)×50=50 ml. CalculateV_(GFP)=(n_(GFP))×(dose)×F=20×(0.05)×50=50 ml.

Calculate the number of plates (P) of 293T cells needed for each virususing the equation: (P)×(Y)=V, where V is V_(OS), V_(CK), V_(NL) orV_(GFP) (from calculation above) and Y is the yield of supernatant for agiven plate format. See Table below for Y-values. If the calculation forP is a non-integer, round up to the nearest integer. Prepare an excessnumber of 293T plates if necessary.

Solve the equation P_(OS)=V_(OS)÷ (Y) For the example above: V_(OS)=100ml, choose the 15 cm format for larger yields per plate, thus Y=14.5.P_(OS)=100÷ 14.5=6.8 plates. Round up to P_(OS)=7 plates.V_(CK)=V_(NL)=V_(GFP)=50 ml, thus solving the equation for P_(CK),P_(NL), or P_(GFP): 50÷ 14.5=3.4 plates. Round up to 4 plates each.P_(CK)=P_(NL)=P_(GFP)=4 plates.

Seeding Viral Supernatant 293T density Yield (Y) Media 10 cm plate   5 ×10⁶  4.5 ml D10F 15 cm plate 13.5 × 10⁶ 14.5 ml D10F

Wash the 293T cells with PBS, and add enough trypsin to cover themonolayer. Incubate at room temperature for 10 minutes, then dislodgethe cells by rapping the side of the dish. Collect the cells in a 50 mltube(s). Wash each plate with a small volume of D10F. Collect andcombine the wash media and cells. Mix thoroughly and transfer a 300 ulaliquot to a Cedex cup and count the cells. Alternatively use TrypanBlue and a hemacytometer. Centrifuge at 350×g for 10 minutes. Aspiratethe supernatant and resuspend the pellet in fresh D10F. Calculate(P_(OS)+P_(CK)+P_(NL)+P_(GFP)), the total number of plates from the stepof calculation of the number of plates (P) of 293T cells needed for eachvirus. Using the seeding densities in the table (below), seed therequired number of 293T cells to each plate. Incubate in D10F forapproximately 24 hours at 37° C./5% CO₂. Transfection efficiency andthus virus production is reduced if the cultures are over- orunder-confluent. Visualize the cells under the microscope to ensure thatconfluency is optimal (approximately 90-95%).

Calculations are made to assess how much of each MMLV or control plasmid(μg_(OS), μg_(CK), μg_(NL), μg_(GFP)) or is needed for transfection ofeach set of 293T cell plates. To simplify the calculations, it isrecommended that the concentration of each plasmid DNA sample beadjusted to 1.0 or 2.0 mg/ml. Choose the appropriate value from thechart below (see column labeled “Vector”), and multiply by P_(OS)P_(CK), P_(NL) or P_(GFP). Then divide by the plasmid DNA concentration(C_(OS), C_(CK), C_(NL), or C_(GFP)) to determine the required volumes(μl_(OS), μl_(CK), μl_(NL), or μl_(GFP)).

Vector or control 2843 1238 PEI (1 μg/μl) plasmid (Gag/Pol) (NFkB) 2842(VSVG) 10 cm  40 μl 10 μg   3 μg   1 μg   1 μg plate 15 cm 108 μl 27 μg8.1 μg 2.7 μg 2.7 μg plate

Optional: adjust each plasmid DNA concentration (C) to 1 μg/μl.Following the above example, and assuming C=1; P_(OS)=7, thusμg_(OS)=(27 μg)×7=189÷ C_(OS)=189 μl_(OS). Following the same example,P_(CK)=P_(NL)=P_(GFP)=4, thus μg_(CK)=(27×4=108÷ C_(CK)=108 μl_(CK),μg_(NL)=(27 μg)×4=108÷ C_(NL)=108 μl_(NL), μg_(GFP)=(27 μg)×4=108÷C_(GFP)=108 μl_(GFP).

To determine the total amount of each helper plasmid (μg_(GagPol),μg_(NFkB), μg_(vsv)) or transfection reagent (μl_(PEI)) that is requiredfor ALL plates, choose the appropriate value from the chart above(columns labeled Gag/Pol, NFkB, VSVG, or PEI) and multiply by the sumvalue (P_(OS)+P_(CK)+P_(NL)+P_(GFP)). Then divide by the plasmid DNAconcentration (C_(OS), C_(CK), C_(NL), or C_(GFP)) to determine therequired volumes. Following the example,(P_(OS)+P_(CK)+P_(NL)+P_(GFP))=7+4+4+4=19 and assuming C=1; then,μg_(GagPol)=(8.1 μg)×19=153.9÷C_(GagPol)=153.1 μl_(GagPol),μg_(NFkB)=(2.7 μg)×19=51.3÷ C_(NFkB)=51.3 μl_(NFkB), μg_(VSV)=(2.7μg)×19=51.3÷ C_(VSVG)=51.3 μl_(VSV), μl_(PEI)=108 μl×19=2.052 ml.

Transfection in 10 cm Plate Format: Tube 1_(OS): Aliquot (P_(OS)×0.5) mlof OptiMEM, then add (P_(OS)×40) μl PEI drop-wise with mixing. Do nottouch sides. Incubate 5 min at room temperature. Tube 2_(OS): Aliquot(P_(OS)×0.5) ml of OptiMEM to a second tube. Prepare a cocktail of theappropriate ratio (10:3:3:1) of plasmids. Choose the appropriate values(μg_(OS), μg_(GagPol), μg_(NFkB), and μg_(VSVG)) from the chart above,and multiply by P_(OS) to obtain the required plasmid amounts. Thendivide by C_(OS), C_(GagPol), C_(NFkB), or C_(VSVG) to determine therequired volumes (μl_(OS), μl_(GagPol), μl_(NFkB), and μl_(VSVG)). Addthese volumes to Tube 2_(OS): μl_(OS)+μl_(GagPol)+μl_(NFkB)+μl_(VSVG)and mix. Repeat these steps by substituting the NL or CK or Sox2-GFPplasmid for the OS plasmid. Prepare a corresponding set of tubes: Tubes1_(NL) and 2_(NL), or Tubes 1_(CK) and 2_(CK) or Tubes 1_(GFP) and2_(GFP). Substitute the appropriate P and C values to calculate theappropriate volumes for the Tube 2 cocktail. To make DNA/PEI mixture,combine each Tube #1 with the corresponding Tube #2, mix, and incubateat RT for 20 min. Wash each plate of 293 Ts twice with 5 ml PBS. Add 4ml OptiMEM to each plate. Add 1 ml of the plasmid DNA/PEI mixturedropwise directly to each plate. Incubate 4-6 hours at 37° C./5% CO₂Aspirate the media, then wash each plate with 5 ml PBS. Add 5 ml ofD10F+50 mM HEPES media to each plate. Incubate at 37° C./5% CO₂overnight. Transfer the Sox2-GFP-infected (control) cells to thefluorescent microscope. The fluorescence should be detectable. Incubateat 37° C./5% CO₂ for an additional 24 hours.

Transfection in 15 Cm Plate Format: Tube 1_(OS): Aliquot (P_(OS)×1.0) mlof OptiMEM, then add (P_(OS)×108) μl PEI drop-wise with mixing. Do nottouch sides. Incubate 5 min at room temperature. Following the example,P_(OS)=7: Aliquot 7 ml of OptiMEM to Tube 1_(OS) and add 1.96 ml PEI.Tube 2_(OS): Aliquot (P_(OS)×1.0) ml of OptiMEM to a second tube.Following the example, P_(OS)=7: Aliquot 7 ml of OptiMEM to Tube 2_(OS).Prepare a cocktail of the appropriate ratio (10:3:3:1) of plasmids.Choose the appropriate values (μg_(OS), μg_(GagPol), μg_(NFkB) andμg_(VSVG)) from the chart above, and multiply by P_(OS) to obtain therequired plasmid amounts. Then divide by C_(OS), C_(GagPol), C_(NFkB),or C_(VSVG) to determine the required volumes (μl_(OS), μl_(GagPol),μl_(NFkB), and μl_(VSVG)). Add these volumes to Tube 2_(OS):μl_(OS)+μl_(GagPol)+μl_(NFkB)+μl_(VSVG) and mix.

Following the example, P_(OS)=7 and assuming C=1 for all plasmids:μg_(OS)=27 μg×7 plates=108÷ C_(OS)=108 μl_(OS), μl_(GagPol)=8.1×7=56.7÷C_(GagPol)=56.7 μl_(GagPol), μg_(NFkB)=2.7×7=18.9÷ C_(NFkB)=18.9μl_(NFkB), μg_(VSVG)=2.7×7=18.9÷ C_(NFkB)=18.9 μl_(VSVG). Add thesevolumes to Tube 2_(OS) and mix. Repeat Steps these steps by substitutingthe NL or CK or Sox2-GFP plasmid for the OS plasmid. Prepare acorresponding set of tubes: Tubes 1 and 2_(NL), or Tubes 1_(CK) and2_(CK) or Tubes 1_(GFP) and 2_(GFP). Substitute the appropriate P and Cvalues to calculate the appropriate volumes for the Tube 2 cocktail. Tomake DNA/PEI mixture, combine each Tube #1 with the corresponding Tube#2, mix, and incubate at room temperature (RT) for 20 min. Wash eachplate twice with 10 ml PBS. Add 13 ml OptiMEM to each plate. Add 2 ml ofthe plasmid DNA/PEI mixture dropwise directly to each plate. Incubate4-6 hours at 37° C./5% CO₂. Aspirate the media, then wash each platewith 15 ml PBS. Add 15 ml of D10F+50 mM HEPES media to each plate.Incubate 37° C./5% overnight. Transfer the Sox2-GFP-infected (control)cells to the fluorescent microscope. The fluorescence should bedetectable. Incubate at 37° C. for an additional 24 hours.

Harvesting the Virus Supernatant. Transfer the Sox2-GFP-transduced cellsto the fluorescent microscope again. The majority of cells should beemitting green fluorescence and the fluorescence should be uniformacross the plate. Virus producing cells should also exhibit a noticeablechange in cell morphology. Pool the virus containing supernatant mediafrom each set of transfected cells. (Caution: supernatants containinfectious virus) Filter the virus supernatant through a 0.45 um or 0.8um filter to remove cells and debris. (Note: use cellulose acetate orPES low protein binding filters. Do not use nitrocellulose filters.)MMLV has a limited shelf-life; store the viral supernatants at 4° C. forno more than 4 days. Optional: the supernatants may be stored at −80°C., however the freeze thaw cycle will cause a loss of functionalactivity. Proceed immediately to assess the virus titer using at leastone of the following metrics: a) functional activity on proliferatingJurkat cells or T-cells and/or b) quantitation of viral RNA present perml of supernatant. Quality control assay of MMLV vectors are describedbelow.

To achieve high transduction efficiency of T-cells in 96-well format, itis important to concentrate the virus. However, the concentrated virusis also unstable. Furthermore the window of time in which the T-cellcultures are most highly proliferative (and thus most easily infected)is narrow. It is thus important to coordinate the preparation of thetarget cells and the concentration step. When QC assay(s) have beensatisfied, proceed to activate the target PBMC's T-cells, andconcentrate the virus supernatants for reprogramming

4. Perform Quality Control Assays for Virus Activity:

This protocol describes methods to assess transduction efficiency bytransduction of cells with the following MMLV vectors: Oct4-Sox2 andc-Myc-Klf4, or Oct4-Sox2 and Nanog-Lin28, or a control Sox2-GFP vector.These assays are intended to be used to facilitate derivation of iPScells.

Quality control assay for virus activity: Note: because of the relativeinstability of the virus it is important to be prepared to initiate one(or all) of the QC assays below on the day that the viral supernatantare collected. Virus may be stored at −70° C., however the freeze thawcycle and/or storage of >3 weeks causes a loss of activity. Afterobtaining an acceptable QC assay result the PBMCs should be re-animated.

Perform quantitative real time RT-PCR using aliquots of each viralsupernatant, according to manufacturer's protocol (Clontech).Alternatively, (or additionally), collect proliferating Jurkat cells andcount using the Cedex. Resuspend the cells at 1×10⁶/ml in R10Fcontaining 4 ug/ml polybrene. Seed 100 ul of cells per well to a 96-wellplate. Add 50 ul of virus to three wells and titrate the virus by serialdilution across several rows of the plate. Incubate 48 hours, thencollect cells for FACS analysis. See procedure for intracellularimmunolabeling of Oct4 and flow cytometry. Alternatively (oradditionally) collect infected Jurkat cells for semi-quantitative PCRanalysis. If the virus prep passes QC, proceed to the next step fortransduction of T-cells.

5. Re-animate Donor PBMCs and Activate T-cells:

Efficient reprogramming of human T-cells can be achieved with MMLVvectors only if the production and delivery of the virus supernatantsare carefully coordinated with the activation of the target cells. Hereis disclosed successful activation as a cytokine-induced burst in theproliferation of CD3⁺ cells from a mixed population of PBMCs yieldingthe formation of macroscopic “blast” colonies between 48 and 72 hours inculture. To utilize this activation protocol with MMLV-basedreprogramming vectors, it is important to note that MMLV supernatantsare unstable. Thus the virus should be prepared on a tightly controlledschedule so that fresh virus may be applied to the T-cell culture oneday before blast colony formation. This protocol describes there-animation of cells cryopreserved as described above, and theinduction of blast colonies. Alternative sources of PBMCs may beutilized.

Prepare Media and Cytokines. Prior to the addition of virus, the cellsmust be activated for 48 hours. Thus this step is designated as Day −2.Reprogramming begins on Day 0. Add a working concentration ofPen/Strep/glutamine to AIM-V media. Store at 4° C. for no more than twoweeks. It is recommended that small volume aliquots of IL2 are preparedand stored at −20° C. Thaw one aliquot for use here. After thawing onealiquot, store it at 4° C. for no more than two weeks. OKT3 (1 mg/mlanti-CD3) should be stored at 4° C. Dilute 1 μl in 1 ml of AIMV media toa 1 μg/ml intermediate dilution.

Re-animation of Donor PBMCs and Activation of T-Cells. The day that thePBMCs are thawed is referred to as Day −2. Remove the PBMCs from storageand thaw rapidly in a 37° C. water bath. Dilute the cells (and freezingmedia) with an equal volume of warm RPMI media. Mix gently and transferto a 15 ml tube. Slowly dilute with RPMI to a total volume of 10 ml. Mixthoroughly, remove a 300 μl aliquot, and count the cells using a Cedexalgorithm with a size threshold of 1 micron. Alternatively, stain cellswith Trypan Blue and count with a hemacytometer. Note: it is not unusualto lose 50% of the cells that were present in the primary PBMC sample(prior to cryopreservation). However, the remaining cells should be >90%viable. Centrifuge the cells at 350×g for 10 min, aspirate thesupernatant and resuspend in warm AIM-V+Pen/Strep/glutamine at a densityof 2×10⁶ viable cells/ml. Add 300 IU/ml IL2 and 10 ng/ml OKT3 antibody.Mix the cells, and dispense 100 μl per well in a flat bottom 96-welltissue culture plate, incubate at 37° C., 5% CO₂. Avoid using theperimeter wells if possible, as evaporation is more noticeable in thesewells. Forty eight hours later (Day 0), observe the cells by brightfield microscopy using a 20× objective (or higher magnification). Note:evidence of cell division and clusters of cells (nascent blast colonyformation) should be detectable.

6. Concentrate the Virus Supernatants:

This protocol describes two separate methods for increasing the titer ofMMLV vectors by concentrating retroviral supernatants collected from293T cells following transfection with a combination of reprogrammingvectors (Oct4-Sox2 with cMyc-Klf4 or Nanog-Lin28; representative vectormaps are shown in FIGS. 11A-11C). It is intended to facilitatederivation of iPS cells by retroviral transduction of T-cells. It isrecommended that the titer of the virus supernatant be assayed accordingto Quality control assay of MMLV vectors as described above).

For large volumes of virus, the LentiX method is recommended. Thismethod requires an overnight incubation, thus it should be initiated onDay −1. Alternatively, for virus prep's of 30 ml or less, the Amiconmethod may be used on Day 0.

Un-concentrated MMLV supernatants (prepared according to proceduresdescribed above) may be stored at 4° C. for 4 days without significantloss of activity. After concentrating the supernatant using eithermethod (below), the virus should be kept cold (on ice) and used as soonas possible. If the target cells are NOT ready to be infected uponcompletion of this procedure, store the concentrated virus at −80° C.

Concentrating the Virus (on Day −1) by the LentiX method. Note: thismethod is recommended for large scale virus concentration, (supernatantvolumes >30 ml). Transfer the supernatants into 50 ml tubes and add theLenti-X concentrator according to the manufacturers recommendations.Combine 3 volumes of clarified viral supernatant with 1 volume ofLenti-X Concentrator. Mix by gentle inversion. Incubate overnight at 4°C. 18-24 hours later, on Day 0, centrifuge the samples at 1,500×g for 45minutes at 4° C. After centrifugation, an off-white pellet will bevisible. Carefully remove supernatant, taking care not to disturb thepellet. Residual supernatant can be removed with either a pipette tip orby brief centrifugation at 1,500×g. Gently resuspend the pellet in1/50th of the original volume using cold D10F. The pellet may besomewhat sticky at first, but it should go into suspension quickly.Proceed immediately to apply the virus to the target cells.

Concentrating the Virus by Amicon Filtration Method (on Day 0). Use thismethod to concentrate virus supernatant prep's of 30 ml or less. Wash anAmiconY100,000 MW cassette by adding 10 ml of PBS and centrifuging thedevice at 1000×g for 3 minutes or until all the PBS has passed throughthe filter. Apply 15 ml of supernatant virus to the Amicon cassette andspin at 2000×g for 20 minutes. Typically this will result in anapproximate 10-fold concentration (by volume). Spin the sample for anadditional 5-10 minutes to concentrate the virus more. This process maybe repeated to reduce the volume by as much as 50-fold (final volumeapproximately 300 μl). Repeat this process (in parallel) with each viralvector supernatant. Recommendation: Do not attempt to process more thanfour (4) Amicon cassettes at one time. During long delays thesupernatant will passively drip through the cassette and result in anuneven distribution of weight across the opposing rotor arms. This maycause the centrifuge to be unbalanced. Collect the retentates. Proceedimmediately to apply the virus to the target cells.

7. Transduce the Activated T-cells (on Day 0):

This procedure is for transduction of human peripheral bloodT-lymphocytes with concentrated MMLV-based reprogramming vectors. Thisprotocol describes transduction of T-cells in a 96-well plate with theMMLV-based reprogramming vectors Oct4-Sox2, c-Myc-Klf4, or Nanog-Lin28,or Sox2-GFP, or combinations thereof. A quality control assay describedabove is recommended to assess viral activity prior to using thisprotocol.

The day of transduction represents the initiation of the reprogrammingprocess (designated as Day 0). This time point occurred 48 hours afterPBMCs were thawed and activated in 96-well format (as described inProcedure 5. Re-animate donor PBMCs and activate T-cells). ConcentratedMMLV vectors should be prepared in advance according to Procedures 3, 4,and 6).

Observe the cells under phase microscopy. There should be evidence ofnascent blast colony formation.

Optional: Collect cells and count. Typically, the number of PBMCs dropssignificantly within 24 hours of activation, (day −2 to day −1) toapproximately 25-50,000 cells per well. Between 24 and 48 hours (day −1to day 0), the cell number is typically unchanged. Between Day 0 and Day1, the ATP content increases and nascent blast colony formation appears.The cell number on Day 0 is typically between 1 and 2×10⁵ per well.Between Days 0 and 1, blast colonies should be apparent and cell numbersincrease significantly.

Optional: collect cells for FACS analysis to characterize T-cells.Previous trials across multiple PBMC donors show >90% of cells displayanti-CD3 surface labeling on Day 0. The distribution of CD4+ and CD8+cells varies. Typically, there are twice as many CD4+ cells compared toCD8+ cells.

Combine equal volumes of each concentrated virus and add 8 μg/mlpolybrene and 300 units/ml IL-2. Prepare enough of this mixture for thegiven number of wells to be infected.

Following the procedure described in Procedure 3: ten (10) differentdonor T-cell samples were seeded to 7 wells, each in 96-well format andactivated; they occupy a total of 70 wells. Two wells from each donorwill receive OS+CK reprogramming viruses (n_(OS+CK)=20). Two wells willreceive OS+NL reprogramming viruses (n_(OS+NL)=20). Two wells willreceive a control Sox2-GFP virus (n_(GFP)=20). The one remaining wellwill represent a non-transduced control. Combine (50 μl_(OS)+50μl_(CK))×n_(OS+CK)=2 ml; add 2 μl of polybrene and 1.2 μl of IL-2.

Combine (50 μl_(OS)+50 μl_(NL))×n_(OS+NL)=2 ml; add 2 μl of polybreneand 1.2 μl of IL-2. Combine (50 μl_(GFP)+50 D10F)×n_(GFP)=2 ml; add 2 μlof polybrene and 1.2 μl of IL-2. Combine 2 ml D10F, 2 μl of polybreneand 1.2 μl of IL-2 for mock-infections.

To undisturbed wells, add 100 μl of the virus cocktail to each well. Mixthe cells gently with the pipettor. Perform a mock-infection by adding100 μl of D10F+300 IU/ml IL2+8 μg/ml polybrene. Mix the cells gentlywith the pipettor. Centrifuge the 96-well plate for 90 minutes at 1000×gat 32° C. using the appropriate biocontainment adapters. Transfer theplate to the incubator at 37° C./5% CO₂ overnight.

Plate Irradiated MEFs (DAY 0, or DAY 1) in preparation for reprogrammingby MEF-co-culture (according to Procedure 8).

On DAY 1—24 hours after the initial exposure to virus, inspect the cellmorphology. Blast colonies should be plainly visible under themicroscope. Optional: Collect cells, centrifuge, resuspend in(virus-free) D10F media and count on the Cedex. Alternatively use TrypanBlue and a hemacytometer.

Carefully remove 100 μl of media from each well without disturbing thecells. For multiple wells, use a multi-channel pipettor, being carefulnot to lower the tips too close to the bottom of each well. Discard thismedia in a beaker or tray containing 10% bleach. Replace the media with100 ul of fresh D10F+HEPES+IL2 (300 u/ml). The following day, repeat themedia removal steps (Day 2).

Verify expansion of T-cells and assess transduction efficiency (DAY 2)according to Procedure 9 described below.

8. Plate Irradiated MEFs (on Day 0 or Day 1):

This section describes a method for plating mouse embryonic fibroblasts(MEFs) on gelatin coated wells, which is intended to facilitatederivation of iPS cells.

Plate Irradiated MEFs for the production of conditioned media (CM).Order MEFs 2-3 days before intended use. Following the example inProcedure 3 for T-cell reprogramming, calculate the amount of MEF-CMnecessary to maintain reprogramming co-cultures for approximately 20“feedings” in 6-well format. Each feeding requires removal andreplacement of 1.25 ml per well.

10 donor samples (transduced T-cells)×two experimental conditions (SO+CKvs. SO+NL)×three wells per condition=60 wells was used. SO refers to abicistronic vector having Sox2 and Oct4, CK refers to bicistronic vectorhaving cMyc and Klf4, and NL refers to Nanog and Lin28, all without anyfluorescent marker (vector maps are represented in FIGS. 11A-11C).Calculate the volume of MEF-CM needed. (60×20×1.25 ml=1.5 liters).

Calculate the number of T75 flask-MEF cultures required to generate asufficient volume of MEF-CM. (Note: repeated collections from one flaskwill generate approximately 120 ml of MEF-CM.). Following the exampleabove, to generate 1.5 liters of MEF-CM: 1500 ml÷ 120 ml/flask=12.5flasks. Round up to 13 flasks.

Add 12 ml of sterile 0.1% gelatin per T75 flask. Incubate for at least 1hour in the incubator (37° C./5% CO₂). Aspirate gelatin and add 20 ml ofhigh density irradiated MEFs (˜2.1×10⁵ cells/ml). Incubate overnight(37° C./5% CO₂). Visualize cells to ensure that MEFs have becomeattached. 24 hours after plating, aspirate the media and replace with 20ml of hES media per flask. 24 hours later, collect ˜20 ml of MEF-CM fromeach flask. Repeat steps 6.8 and 6.9 for five additional days. FreezeMEF-CM at −20° C. (Add 4 ng/ml zbFGF and filter only before using forIPS culture then filter.

Plate Irradiated MEFs (DAY 0, or DAY 1) for reprogramming co-cultures.Order MEFs 2-3 days before intended use. MEFs should be plated 1 or 2days prior to adding the transduced cells for reprogramming co-cultures.Following the example in Procedure 3 for T-cell reprogramming, calculatethe number of wells of MEFs necessary to receive 10 donor samples×3wells per donor×3 experimental conditions (SO+CK versus SO+NL versus GFPcontrols)^(,) 90 wells. Calculate the number of 6-well plates needed.(90÷ 6=15 plates). Add 2 ml of sterile 0.1% gelatin per well (6-wellformat). Optional: Coat 96-well plates with 100 μl gelatin per well.Incubate for at least 1 hour in the incubator (37° C./5% CO₂). Aspirategelatin and add 2.5 ml of irradiated MEF cell suspension onto each well(6-well format). Optional: For 96-well format, aspirate gelatin and add200 μl of MEFs cell suspension onto each well. Incubate overnight (37°C./5% CO₂). Visualize cells to ensure that MEFs have become attached.

9. Perform Quality Control Assays to Assess Transduction Efficiency (onDay 2):

This procedure describes quality control assays to assess MMLVtransduction of human peripheral blood T-cells. The assays are intendedto detect the presence of transgenes or reprogramming factors present intargeted T-cell populations 48 hours after the cells are exposed toconcentrated MMLV vectors comprising combinations of Oct4-Sox2,c-Myc-Klf4, Oct4-Sox2 and Nanog-Lin28.

Activate human T-cells according to procedures described above. 48 hourslater, infect the activated T-cells according to Procedure 7. Count thecells using the Cedex, or a hemacytometer.

Following the example in procedure 3 (and continued in procedure 7),remove 1000 of supernatant media from each well without disturbing theactivated cells. All wells should have approximately 100 μl remaining.Mix and collect the remaining 100 μl of cells from one of the twoSox2-GFP-infected wells and transfer the cells directly into a Cedexcup. Wash each well with 200 μl of PBS; collect and combine the washinto the Cedex cup. Adjust the final volume to 300 μl if necessary.Repeat mix and wash steps for each of the 10 donor T-cell samples.

Count the cells on the Cedex using the T-cell algorithm (sizethreshold=1 micron). Record the cell density (Note: there should be2-4×10⁵ per well). Alternatively, thoroughly mix the cells from one welland remove 100 and mix with 10 μl of trypan blue, then count cells on ahemacytometer. (Note: this counting method is less accurate than theCedex; however, it uses less cells.)

Assess the Transduction Efficiency of the Transduced T-Cells. Optional:use the fluorescent microscope to visualize the cells that weretransduced with the Sox2-GFP virus. With a multi-channel pipetor, mixand collect the remaining 100 μl of cells from one well ofSox2-GFP-infected T-cells from each donor sample (10 wells). Transferthe cells into a corresponding set of wells in a 96-well V-bottomcollection plate. Mix and collect the remaining 100 μl of cells from thecontrol (non-infected) wells from each donor sample (10 wells). Washeach well with 75 μl of PBS; collect and combine the wash into thecorresponding wells of the collection plate. Centrifuge the collectionplate for 10 minutes at 350×g. With a multi-channel pipetor, carefullyremove the supernatants without disturbing the pellet in each well.Discard the supernatants in a beaker or tray containing 10% bleach.Resuspend and wash the pellets with 150 μl FACS buffer per well.Centrifuge the collection plate for 10 minutes at 350×g. Carefullyremove the supernatants without disturbing the pellet in each well.Resuspend the pellets in FACS buffer containing 2-5 μg/ml anti-CD3-APC.Incubate at room temperature in the dark for 45 minutes.

Centrifuge the collection plate for 10 minutes at 350×g. Carefullyremove the supernatants without disturbing the pellet in each well.Resuspend and wash the pellets with 150 μl FACS buffer per well. Repeatcentrifuge steps.

Resuspend and wash the pellets with 100 μl FACS buffer containing 1μg/ml propidium iodide per well. Analyze the cells with the Accuri.Assess transduction efficiency by estimating the percentage of livecells that express GFP⁺. Assess the percentage of GFP⁺ cells that areCD3+ by gating on the GFP⁺ population.

10. Co-culture Transduced T-cells and MEFs (Days 3-30):

This procedure describes a procedure for co-culturing human T-cellstransduced with reprogramming factors on mouse embryonic fibroblasts(MEFs). The protocol describes the co-culture of transduced T-cells onadherent MEFs and methods for re-feeding these cells.

1. Prepare MEF-conditioned media according to Procedure 8. Prepare thisreagent before proceeding to Step 4 below.

2. Plate Irradiated MEFs for Reprogramming (according to Procedure 8).Receive and plate 2.5 ml of MEFs per well (in 6 well format) or 200 μlper well (in 96 well format). 24-72 hours later, replace the media with2 ml of hES:D10F media (for 6 well format) or 100 μl per well (for 96well format).

3. Perform quality control assays to assess the transduction efficiency(see Document #100405.RDL.09) two days after T-cells have been exposedto retrovirus. For T-cell reprogramming, this time point is designatedas Day 2. If the transduction efficiency is adequate, proceed to Step 4.

4. Collect transduced T-cells (according to Procedure 7) and confirmthat activation and transduction were successful according to Procedure.

For 6-well MEF plates: transfer 0.5-4×105 cells (in a volume of 25-100ul) per well. Add dropwise across the entire surface. Optional: titratethe input cell number across 3 wells on the MEF plate. For 96-well MEFplates: transfer 1-8×10⁴ cells (in a volume of 10-25 ul) per well.(Note: avoid using the perimeter wells if possible.) Optional: titratethe input cell# across multiple wells on the MEF plate.

The example below follows that described in Doc#100405_RDL_03; there are10 sets of activated T-cell cultures derived from 10 blood donorsamples. Each donor sample was arrayed across seven (7) wells of a96-well plate and T-cells were activated. Two wells (2) were infectedwith SO+CK MMLV vectors; two wells (2) were infected with SO+NL MMLVvectors.

From each donor, collect the SO+CK infected T-cells and combine thecells in a FACS tube or 15 ml conical tube. From each donor, collectSO+NL infected T-cells and combine them in separate tubes. Optional: toprecisely account for the seeding density (i.e. how many T-cells aredelivered to the MEF plates), mix cells, remove 10 μl aliquots, combinewith 10 μl Trypan blue and count cells with a hemacytometer. Centrifugethe samples at 350×g for 10 minutes using the appropriate biocontainmentdevices and centrifuge adapters. Carefully remove the supernatant anddiscard this media in a beaker or tray containing 10% bleach. Resuspendthe cells with 400 μl of hES:D10F (approximately 4-8×10⁵ cells total).Mix and transfer 200 μl of cells (2-4×10⁵ cells) dropwise to one well ona MEF plate (6-well format).

Dilute the remaining cells 2-fold with hES:D10F media, then transfer 200μl of cells (1-2×10⁵ cells) dropwise to a second well on the same MEFplate. Dilute the remaining cells 2-fold with hES:D10F media, thentransfer 200 μl of cells (approximately 0.5-1×10⁵ cells) dropwise to athird well on the same MEF plate. Incubate overnight at 37°/5% CO₂.

5. Maintenance: Feeding Schedule (Day 4-Day 30). Two days later (on day4), replace 50% of the media from each well with hES media+100 ng/ml ofzebrafish FGF (growth media) using the following method: Remove the6-well plates from the incubator. Stand one side of the plate on adiscarded/unused 10 cm-culture dish lid, allowing the culture media toflow to one side of the well. (Note: at this angle, the media shouldstill be covering the entire monolayer of MEFs and not spilling out ofthe well). Let the cells settle for 10 minutes. Remove each lid from theMEF plate, and carefully/slowly aspirate 50% of the media from thesurface of the culture. Be careful not to aspirate cells. Keep thelid(s) for all subsequent feedings. Optional: Collect the aspirate,centrifuge 350×g for 4 minutes, resuspend in 1 ml media and count onCedex. Verify that you are losing less than 1% of the cells. Add 1.25 mlof fresh growth media dropwise in a circular motion trying not todisturb the cells. Return the plate to the incubator. To replace themedia in a 96-well plate co-culture, use a multi-channel pipettor andinsert the tips approximately half way to the bottom of the wells.Slowly aspirate 100 ul from the surface of the culture. Be careful notto aspirate cells. (Note: compared to the 6-well co-culture format, theT-cells will appear more stationary since the media is not easilyagitated in this format.) Add 100 ul of fresh media (hES media+100 ng/mlof zFGF) dropwise trying not to disturb cells. Return the plate to theincubator.

On Day 6, repeat the feeding method on Day 4 described above. On Day 8,re-feed the cells (as above) with growth media+20% MEF-conditionedmedia. Repeat the feeding step on Day 8 every 48 hours. Visually inspectthe wells during this time period to monitor colony formation.

11. Identify and Pick Colonies Expressing Tra1-60:

The procedure describes a guidelines for identifying and pickingTra1-60⁺ colonies grown on MEF co-cultures under reprogrammingconditions. Under the appropriate conditions, colonies of iPS cells willarise following the introduction of reprogramming factors into primaryhuman cells. This protocol utilizes an antibody to Tra1-60 tofluorescently label putative iPSC colonies that arise in MEFco-cultures. By comparing the fluorescent labeling pattern and themorphology of the colonies, colonies are assigned a score which is aqualitative measure of pluripotency. This scoring system facilitatesselective expansion of putative iPS cells for further characterization.

Following transduction with reprogramming factors, human cells areco-cultured on MEFs for 15-30 days. During this period of time, thecells should be visually inspected for colony formation. When coloniesare visible, but not overgrown, count or estimate the total number ofcolonies present.

If the reprogramming vector(s) do NOT include a fluorescent reporter, gothe following step: one or two days before picking colonies, seedirradiated MEFs on to a set of gelatin-coated 6 well plates or 96-wellplates (according to Procedure 8) or 10 cm dishes.

If the reprogramming vector(s) include a fluorescent reporter, nascentcolonies should be monitored under the fluorescent microscope. Make noteof fluorescent and non-fluorescent colonies. Reprogramming eventstypically silence fluorescent reporters. However, in some cases, thecells may remain fluorescent. For this reason it is important to avoidusing a fluorescent antibody (below) with fluorescent spectra thatoverlaps with that of the reporter.

Carry out the anti-Tra1-60 live cell labeling protocol, as follows: Washwells to be stained with DMEM/F12 (serum-free) media two times. Diluteprimary antibody in growth medium (hES) to the working dilution. Filterthe diluted antibody using a 0.22 um sterile filter. Add diluted primaryantibody to the cells. Add a sufficient volume to cover the monolayer.Incubate at 37° C. and 5% CO₂ for 45 min-1 hr. Wash the cells withDMEM/F12 media two times. Note: If you use a fluorescently labeledprimary antibody, replace media with fresh hES and image the cells.Otherwise: Dilute the secondary antibody in growth medium (hES). Filterthe diluted secondary antibody using a 0.22 um sterile filter. Adddiluted antibody to the cells. Incubate at 37° C. and 5% CO₂ for 30 min.Wash cells once with DMEM/F12 and replace the media fresh hES+CM and usethe fluorescent microscope to take images.

If there are numerous colonies, assign scores after acquiring andobserving digital images. For only a few colonies, score each colonywhile the samples are under the microscope. Assign a “morphology” scoreusing bright field microscopy, according to the descriptions below:1=the colony would be described as partially reprogrammed; the colonyhas a diffuse border, and/or differentiated cells at the border; and/ordifferentiated cells that are fibroblast-like with discernable cytoplasmand nuclei. 2=the colony is distinct enough to be picked manually; thismay comprise a colony of non-differentiated cells (with lowcytoplasmic:nuclei ratio) with a semi-contiguous tight borderinterrupted by differentiated areas, or a colony with a completelycontiguous tight border surrounded by a halo of differentiated cells (a“fried egg” morphology). 3=a colony with classic morphology; tightborders; free of differentiated cells, cells have low cytoplasmic:nucleiratio.

Assign a “Tra1-60” score while visualizing each colony underfluorescence microscopy, according to the descriptions below: 0=nolabeling; 1=weak or spotty; 2=heterogeneous or irregular labelingpattern; little or no evidence of a defined border; 3=uniform labelingwith defined border.

Identify and Pick Tra1-60-positive colonies Mark the plate with ink toidentify colonies with the highest score. A score of “3-3” is ideal,however, there is precedence for picking and successfully propagatingcolonies with less than ideal morphology (e.g., those that might bescored as “2-3” or “2-2”).

To pick colonies from a 6-well plate (or 10-cm dish), transfer the cellsto a picking hood. While visualizing the colony with a dissectingmicroscope, manually draw a pipet tip across the surface of the dish (ina “tic tac toe board” fashion) around the colony border. This actionshould break the colony up into 3-6 pieces, freeing it from thesurrounding MEFs. Draw the colony fragments into the pipet tip. (Note:Be aware that dislodged fragments that remain in the original well willlikely re-attach and produce secondary colonies. This may confound yourcolony counts and estimates of reprogramming efficiency). Transfer thefragments directly into a recipient well of a 6 well plate containingMEFs with hES media+100 ng/ml zebrafish bFGF (growth media).

To pick colonies from 96-well format, identify wells with only a singlecolony with good morphology and Tra1-60 labeling scores. Aspirate themedia from the wells. Add dispase and incubate for 7 minutes at 37° C.Dislodge the colony by gently pipetting up and down. Transfer the colonyfragments in a 15 ml tube and dilute with hES media. Centrifuge at 350×gfor 10 min. Aspirate the supernatant, then resuspend in growth media.

Transfer the fragments directly into a recipient well of a 6 well platecontaining MEFs in growth media. The colony fragments should attach tothe new MEF and form multiple new colonies. 24 hours later, replace themedia with fresh growth media. Monitor the proliferation, and morphologyfor until the cells become confluent. Replace the media each day withfresh growth media.

EXAMPLE 10 Materials

Materials used in Examples 1-9 are shown in Tables 5-7.

TABLE 5 Reagents Catalog Reagent Supplier Number DMEM Invitrogen 11965DMEM/F12 Invitrogen 11330 FBS Hyclone SH30070.03 AIM-V Invitrogen12055-091 Pen/Strep/L-Glutamine Invitrogen 10378-016 Opti-MEM Invitrogen31985 KOSR Invitrogen 10828 NEAA Invitrogen 11140 B-mercaptoethanolInvitrogen M7522 (BME) L-glutamine Invitrogen 21051-024 zbFGF in housenone rhIL2 Peprotech 200-02 OKT3 (anti-CD3) eBiosciences 16-0037-81Functional Grade High density irradiated Wi-Cell none MEF for CMIrradiated MEF Wi-Cell none PBMCs via Biological 213-14-04 leukophoresisor freshly Specialty isolated blood sample Corporation 1M HEPESInvitrogen 15630 0.05% Trypsin/EDTA Invitrogen 25300 PBS (Ca and Mgfree) Invitrogen 14190 Gelatin Sigma C1890 Trypan Blue Stain Invitrogen15250-061 PEI Sigma 03880 10 cm tissue culture Falcon 353003 plates 6well tissue culture Nunc 140685 plates 250 ml media filters Nunc 568-002

TABLE 6 FACS D10F Buffer DMEM 90% PBS (Ca and Mg free) FBS 10% 2% + or −HEPES 50 mM (0.1% NaN3 optional)

TABLE 7 Conditioned Media (CM) DMEM/F12 80% KOSR 20% NEAA  1% BME  0.1mM L-glutamine  1 mM zbFGF 100 ng/ml Expose to high density MEF culture.Collect medium daily, for 8-10 days. Add zbFGF only before using for IPSculture then filter.

EXAMPLE 11 Generation of iPS Cells from CD34⁺ Hematopoietic Cells

PBMCs were isolated from leukopak or freshly drawn blood samples asdescribed in Example 1. MACS separation for CD34⁺ cells was performedusing a Indirect CD34 MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach,Germany) or a Direct CD34 MicroBead Kit or a lineage depletion kit(Miltenyi Biotec, Bergisch Gladbach, Germany) according to manufacturersinstructions. A fraction of the CD34⁺ MACS purified cells were collectedfor FACS analysis, and the remaining cells were replated in lowattachment 6 well plates using CD34⁺ cell expansion media, below. TheCD34⁺ cell enrichment can be performed using the CD34 direct microbeadsor the indirect CD34 hapten antibody staining kit.

TABLE 8 CD34 Expansion Media CD34 Exp. Media Concentration Supplier Cat#Stem Pro-34 48 ml Invitrogen 10639-011 Step Pro-34 650 ul InvitrogenSupp Penn/Strep 0.5 ml Invitrogen L-Glut/BME 0.5 ml Invitrogen/ SigmaIL3 20 ng/ml Invitrogen PHC0034 Flt3L 100 ng/ml Invitrogen PHC1705 SCF100 ng/ml Invitrogen PHC2115

CD34⁺ cell expansion media: Stem Pro 34 (Invitrogen) is mixed thedesired volume of nutrient supplement according to the Manufacturer'sinstructions. Stem Pro complete is supplemented with 100 ng/ml ofrecombinant Stem Cell Factor and recombinant 100 ng/ml of Flt-3 Ligandand 20 ng/ml of recombinant human interleukin-3 (IL-3). The medium isalso supplemented with fresh 1% Glutamine and 1% Penicillin Streptomycinsolution, All the supplements were mixed and the media was filteredbefore use.

Cells were seeded approximately 24 hours prior to transfection, via themethod described above. 293T cells were also transfected for retrovirusproduction, and hematopoietic cells were then transfected with either(OCT4, SOX2, NANOG, and Lin28) or (OCT4, SOX2, KLF4, c-MYC) genes,delivered by MMLV retroviruses, as described above. As a result of theseexperiments, it was observed that CD34⁺ hematopoietic cells transfectedwith either set of the above genes resulted in the generation of new iPScell lines.

The following protocol was used for reprogramming PBMCs using MMLVretroviruses: Place MACS LS separation column at −20° C. for quickcooling (Alternately the columns and MACs buffer can be cooled overnightat 4° C.). Thaw appropriate number of PBMC vials to collect ˜3×10⁸cells. Bring cells up to 5 ml with MACS Buffer (keep buffer coldthroughout procedure). Centrifuge at 1200 rpm×5 min, aspiratesupernatant and resuspend in MACS Buffer. Count cells usinghemacytometer. Divide cell suspension into 3 tubes of 1×10⁸ andcentrifuge 300×g for 10 minutes. The CD34⁺ cell enrichment can beperformed using the CD34 direct microbeads or the indirect CD34 haptenantibody staining kit. Resuspend each cell pellet in 300 ul of MACSBuffer. Add 100 ul of FcR Blocking Reagent per tube, mix. Add 100 ul ofCD34-Hapten-Antibody or direct CD34 beads per tube, mix. Incubate at 4°C. for 15 minutes. Wash cell with 5 ml of MACS Buffer and centrifuge300×g for 10 minutes. Aspirate supernatant completely. Resuspend thecells in 500 ul of MACS Buffer. The cells are ready separation if usingthe one step CD34 direct microbeads. If using the indirect CD34separation beads then there is one more incubation step with theanti-hapten micorbeads before separation. Add 100 ul of Anti-HaptenMicrobeads, mix. Incubate at 4° C. for 15 minutes. Wash cells with 2 mlof MACS Buffer, centrifuge 300×g for 10 minutes. Resuspend in 500 ulMACS Buffer. Remove MACS LS Columns from 4° C. Place column on separatormagnet. Rinse column with 3 ml of MACS Buffer. Apply cell suspensiononto the column. Collect unlabeled cells that pass through and washcolumn with 3 ml of MACS Buffer. Repeat wash 2 additional times. Removethe column from the magnet and place in a suitable collection tube. Add3 ml of MACS Buffer and collect enriched CD34⁺ cell fraction by flushingout the column with plunger provided. Collect a fraction of enrichedpopulation for FACS analysis. Replate the remaining cells in lowattachment 6 well plate using CD34⁺ cell expansion media. When using thelineage Cell Depletion kit the cells are incubated with a biotinylatedcocktail of lineage positive antibodies (CD2, CD3, CD11b, CD14, CD15,CD16, CD19, CD56, CD123, CD235a) to remove mature hematopoietic celltypes such as T cells, B cells, NK cells, dendritic cells, monocytes,granulocytes, erythroid cells. Following the incubation the cells arewashed and incubated with anti-Biotin micro beads. The cell suspensionis washed and separated manually column or by using the AutoMACs cellseparator.

Identifying and Picking iPS colonies was done via the following method:Morphologically colonies were generally dense and comprised of small,compact cells with enlarged nuclei and 2 distinct nucleoli. Colonies arefrequently too dense to observe such distinct features and appear tohave differentiated material on the center of the colony. Borders ofcolony are usually defined. However, the blood iPS cells (BiPSCs)appeared more diffuse with shaggy boundaries, a feature not typicallyconsistent with previous iPSCs derived from fibroblasts. Colonies willsilence the GFP and RFP expressed from the integrated viral DNA. Somebona fide colonies will lose fluorescence by 20 days post infection andsome have lost fluorescence after they have been picked and transferred˜35-40 days post-infection. All colonies should be lacking GFP and RFPexpression (though some expression was noted in single cells nearby. Topick manually, a pipet tip was used to break it up colonies into 3-6pieces to increase the probability of freeing stem cells from thesurrounding MEF and hematopoietic stem cells. Picking was avoided untilmultiple colonies have formed so as to avoid confounding your counts oftotal colonies, i.e. to avoid the possibility that a small chunk of acolony resettles and could be falsely counted as a new clone. Cells werethen transferred directly into a recipient well of a 6 well platecontaining MEFs with hES media+100 ng/ml zebrafish bFGF. Proliferation,morphology, and loss of fluorescence were monitored for 1-2 weeks to beconfident that clones are indeed fully reprogrammed. Cells were feddaily. After the picked and plated colonies adhere and displaycharacteristic ES-like morphology, colonies were manually picked asdescribed above again onto a new set of 6 well MEF plates, with dailyfeeding. As wells become confluent, passage as normal iPSC line withcollagenase, freeze down aliquots at various passages, and test thaweach set.

The colonies that are picked and expanded are stained for the presenceof pluripotency markers (SSEA-4, Oct3/4, Tra-160, Tra-181). The colonieswere also stained for the presence of alkaline phosphatase activity. Theclones were tested for the presence of a normal karyotype and theidentity of the iPS clones was confirmed to the parental cell type byFISH analysis. The results of these tests indicated that CD34⁺ cells hadbeen successfully converted into iPS cells. It was observed that,although the efficiency of the transfection was higher when CD34⁺ cellswere transfected with (Sox2, Oct4, c-Myc, and Klf-4), iPS cells derivedfrom the transfection of CD34⁺ cells with (Sox2, Oct4, Nanog, and Lin28)factors were observed to be more stable during maintenance of clones onirradiated MEFs and Matrigel. In further experiments, CD34⁺ cellsobtained from leukopak and donor blood were successfully converted intoiPS cells via transfection with (Sox2, Oct4, c-Myc, and Klf-4). Thereprogramming efficiency of the progenitor cell type was observed to beapproximately 10 colonies per 100,000 cells.

***

All of the methods disclosed and claimed herein can be made and executedwithout undue experimentation in light of the present disclosure. Whilethe compositions and methods of this invention have been described interms of preferred embodiments, it will be apparent to those of skill inthe art that variations may be applied to the methods and in the stepsor in the sequence of steps of the method described herein withoutdeparting from the concept, spirit and scope of the invention. Morespecifically, it will be apparent that certain agents which are bothchemically and physiologically related may be substituted for the agentsdescribed herein while the same or similar results would be achieved.All such similar substitutes and modifications apparent to those skilledin the art are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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The invention claimed is:
 1. A method for producing induced human induced pluripotent stem cells from T cells comprising the steps of: (a) obtaining a cell population comprising human T cells; and (b) producing iPS cells from the T cells of the cell population through the introduction of nucleic acids encoding reprogramming factors selected from Sox, Oct, cMyc, Klf4, Nanog, SV40 Large T antigen and Lin28, the nucleic acids being comprised in one or more Epstein-Barr virus (EBV)-based episomal replicating vectors, and culturing the cells under defined, feeder-free conditions to provide a human iPS cell population that is essentially free of integrated, exogenous viral elements.
 2. The method of claim 1, wherein the source of the cell population is a blood sample, blood components, bone marrow, lymph node, fetal liver, or umbilical cord.
 3. The method of claim 2, wherein the source of the cell population is a blood sample of from about 1 to about 5 ml.
 4. The method of claim 3, wherein the source of the cell population is a blood sample of about 3 ml.
 5. The method of claim 1, wherein the source of the cell population comprising T cells is a subject whose cells have not been mobilized with extrinsically applied G-CSF.
 6. The method of claim 1, wherein the cell population has been cryopreserved.
 7. The method of claim 1, wherein the cell population comprising T cells is prepared in vitro or in vivo under conditions that will activate the T cells.
 8. The method of claim 7, wherein a cell population comprising T cells is cultured in the presence of an anti-CD3 antibody.
 9. The method of claim 1, wherein the cell population comprising T cells is cultured in vitro with one or more cytokines to expand the T cell population therein.
 10. The method of claim 9, wherein the one or more cytokines comprises IL-2.
 11. The method of claim 1, wherein the T cells are CD4⁺or CD8⁺T cells.
 12. The method of claim 1, wherein the T cells are T helper 1 (TH1) cells, T helper 2 (TH2) cells, TH17 cells, cytotoxic T cells, regulatory T cells, natural killer T cells, naïve T cells, memory T cells, or gamma delta T cells.
 13. The method of claim 1, wherein the cell population comprises from about 90% to about 99% T cells.
 14. The method of claim 1, wherein the cell population comprises from about 97% to about 99% T cells.
 15. The method of claim 1, wherein the cell population comprises at least 1×10³ T cells.
 16. The method of claim 1, wherein the cell population comprises at least 5×10³ T cells.
 17. The method of claim 1, wherein the cell population comprises from about 1×10⁶ to about 2×10⁶ T cells.
 18. The method of claim 1, wherein the reprogramming factors are reprogramming proteins comprising a Sox family protein and an Oct family protein.
 19. The method of claim 18, wherein one or more of the reprogramming proteins is operatively linked to a protein transduction domain.
 20. The method of claim 17, wherein the reprogramming factors are encoded by one or more expression cassettes and comprise a Sox family protein and an Oct family protein.
 21. The method of claim 20, wherein the reprogramming factors are encoded on one or more expression cassettes that comprise at least one polycistronic transcript unit.
 22. The method of claim 21, wherein the polycistronic transcript unit comprises at least two reprogramming genes.
 23. The method of claim 22, wherein the polycistronic transcript unit comprises a Sox gene and an Oct gene.
 24. The method of claim 22, wherein the polycistronic transcript unit comprises a cMyc gene and a Klf4 gene.
 25. The method of claim 22, wherein the polycistronic transcript unit comprises a Nanog gene and an Lin28 gene.
 26. The method of claim 21, wherein the polycistronic transcript unit comprises a reprogramming gene and a selectable or screenable marker.
 27. The method of claim 21, wherein the polycistronic transcription unit comprises an internal ribosome entry site (IRES) or a sequence coding for at least one protease cleavage site and/or self-cleaving peptide for polycistronic transcription.
 28. The method of either of claim 18 or 20, wherein the Sox family protein is Sox2.
 29. The method of either of claim 18 or 20, wherein the Oct family protein is Oct4.
 30. The method of either of claim 18 or 20, wherein the reprogramming proteins further comprise Nanog, Lin28, c-Myc, Klf4, or Esrrb.
 31. The method of claim 30, wherein the reprogramming proteins further comprise Nanog.
 32. The method of claim 30, wherein the reprogramming proteins further comprise Klf4 and c-Myc.
 33. The method of claim 1, wherein producing iPS cells from the T cells of the population further comprises selecting the iPS cells for one or more characteristics of embryonic stem cells.
 34. The method of claim 33, wherein the characteristic is an adherent property, an undifferentiated morphology, an embryonic stem cell-specific marker or pluripotency.
 35. The method of claim 34, wherein the characteristic is an adherent property.
 36. The method of claim 34, wherein the characteristic is an undifferentiated morphology.
 37. The method of claim 1, wherein the method further comprises differentiating the iPS cells to a differentiated cell.
 38. The method of claim 37, wherein the differentiated cell comprises a cardiomyocyte, a hematopoietic cell, a neuron, a fibroblast or an epidermal cell.
 39. The method of claim 1, wherein the iPS cell population is essentially free of integrated, exogenous viral elements.
 40. A human induced pluripotent stem cell comprising a genome comprising an incomplete set of V, D, and J segments of T cell receptor genes as compared with an embryonic stem cell, wherein the induced pluripotent stem cell is free or essentially free of integrated, exogenous viral elements.
 41. A human induced pluripotent stem cell comprising a genome comprising an incomplete set of V, D, and J segments of T cell receptor genes as compared with an embryonic stem cell, wherein the induced pluripotent stem cell is free or essentially free of integrated, exogenous viral elements and is produced by the process of claim
 1. 